Method for the carry-over protection in DNA amplification systems targeting methylation analysis achieved by a modified pre-treatment of nucleic acids

ABSTRACT

Particular aspects provide methods for specific amplification of template DNA in the presence of potentially contaminating PCR products from previous amplification experiments. Particular embodiments comprise, in a first step, contacting DNA with a bisulfite solution, which sulfonates unmethylated (but not methylated) cytosines, resulting in cytosine deamination and generation of sulfonated uracil. Such sulfonation protects the template nucleic acid from being a target for the enzyme uracil-DNA-glycosylase (UNG), whereas any contaminating DNA, which contains unprotected unsulfonated or desulfonated uracils, is degraded enzymatically while the UNG is active. After UNG treatment and inactivation thereof, the sulfonated uracil bases are converted into uracil by desulfonation. Such aspects have substantial utility for decontamination of nucleic acid samples; e.g., for avoiding amplification of ‘carry over products’ in the context of DNA methylation analysis. In further aspects, the inventive methods can be generally used as simplified methods of bisulfite treatment.

FIELD OF THE INVENTION

Aspects relate generally to nucleic acid amplification reactions andmore particularly to novel compositions and methods for preventingcarry-over contamination within nucleic acid amplification reactionsthat are directed to methylation analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims the benefit of priority to: U.S. ProvisionalPatent Application Ser. No. 60/618,267, filed 12 Oct. 2004 and entitledA METHOD FOR CARRY-OVER PROTECTION IN DNA AMPLIFICATION SYSTEMSTARGETING METHYLATION ANALYSIS ACHIEVED BY A MODIFIED PRE-TREATMENT OFNUCLEIC ACIDS; and to European Patent Application EP 04 090 389.0, filed11 Oct. 2004 of same title, both of which are incorporated by referenceherein in their entirety.

BACKGROUND

In recent decades, molecular biology studies have focused primarily ongenes, the transcription of those genes into RNA, and the translation ofthe RNA into protein. There has been a more limited analysis of theregulatory mechanisms associated with gene control. Gene regulation, forexample, at what stage of development of the individual a gene isactivated or inhibited, and the tissue specific nature of thisregulation is less well understood. However, such regulation can be withthe extent and nature of methylation of the gene or genome. Specificcell types can be correlated with specific methylation patterns, as hasbeen shown for a number of cases (Adorjan et al. (2002) Tumour classprediction and discovery by microarray-based DNA methylation analysis.Nucleic Acids Res. 30 (5) e21).

In higher order eukaryotes, DNA is methylated nearly exclusively atcytosines located 5′ to guanine in the CpG dinucleotide. Thismodification has important regulatory effects on gene expression,especially when involving CpG rich areas, known as CpG islands locatedin the promoter regions of many genes. While almost all gene-associatedislands are protected from methylation on autosomal chromosomes,extensive methylation of CpG islands has been associated withtranscriptional inactivation of selected imprinted genes and genes onthe inactive X-chromosome of females.

Cytosine modification, in form of methylation, contains significantinformation. The identification of 5-methylcytosine in a DNA sequence,as opposed to unmethylated cytosine; that is, the methylation status, isof great importance and warrants further study. However, because5-methylcytosine behaves like cytosine in terms of hybridizationpreference (a property relied on for sequence analysis), itspositions/status can not be identified by a normal sequencing reaction.Furthermore, in any amplification, such as a PCR amplification, thisrelevant epigenetic information, methylated cytosine or unmethylatedcytosine, will be lost completely.

Several methods are known in the art that relate to this problem.Usually genomic DNA is treated with a chemical or enzyme leading to aconversion of the cytosine bases, which consequently allows subsequentbase differentiation. The most common methods are: a) the use ofmethylation sensitive restriction enzymes capable of differentiatingbetween methylated and unmethylated DNA; and b) the treatment with abisulfite reagent. The use of said enzymes is limited due to theselectivity of the restriction enzyme towards a specific recognitionsequence.

Therefore, the specific reaction of bisulfite with cytosine, which, uponsubsequent alkaline hydrolysis is converted to uracil (whereas5-methylcytosine remains unmodified under these conditions) (Shapiro etal. (1970) Nature 227: 1047) is currently the most frequently usedmethod for analyzing DNA for 5-methylcytosine. Uracil corresponds tothymine in its base pairing behaviour; that is, it hybridizes toadenine; whereas 5-methylcytosine does not change its chemicalproperties under this treatment, and therefore still has the basepairing behavior of a cytosine (hybridizing with guanine). Consequently,the original DNA is converted in such a manner that 5-methylcytosine,which originally could not be distinguished from cytosine by itshybridization behavior, can now be detected as the only remainingcytosine using standard molecular biological techniques, for example,amplification and hybridization or sequencing. All of these techniquesare based on base pairing, which can now thereby be more fullyexploited. Comparing the sequences of the DNA with and without bisulfitetreatment allows an easy identification of those cytosines that havebeen unmethylated. An overview of further known methods for detecting5-methylcytosine may be gathered from the following review article:Fraga & Esteller, Biotechniques 33:632, 634, 636-49, 2002.

Again, because the use of methylation-specific enzymes is restricted tocertain sequences (comprising restriction sites), most typical methodsare based on a bisulfite treatment that is conducted before a detectionor amplifying step (for review: DE 100 29 915, A1 p.2, lines 35-46 orthe according translated U.S. application Ser. No. 10/311,661, see alsoWO 2004/067545 ). The term ‘bisulfite treatment’ in this context ismeant to comprise treatment with a bisulfite, a disulfite or ahydrogensulfite solution. As known to a person or ordinary skill in theart (and as used herein), the term “bisulfite” is used interchangeablyfor “hydrogensulfite”.

Several bisulfite-based protocols are known in the art. However, all ofthe described protocols, comprise the following steps: the genomic DNAis isolated, denatured, converted several hours by a concentratedbisulfite solution and finally desulfonated and desalted (see, e.g.,Frommer et al.: A genomic sequencing protocol that yields a positivedisplay of 5-methylcytosine residues in individual DNA strands. ProcNatl Acad Sci USA. 89:1827-31, 1992).

Recent technical improvements of bisulfite methods. The art-recognizedagarose bead method incorporates the DNA to be investigated in anagarose matrix, through which diffusion and renaturation of the DNA isprevented (bisulfite reacts only on single-stranded DNA) and allprecipitation and purification steps are replaced by rapid dialysis(Olek A. et al. A modified and improved method for bisulphite basedcytosine methylation analysis, Nucl. Acids Res. 24, 5064-5066, 1996).

Patent application WO 01/98528 (20040152080) describes a bisulfiteconversion in which the DNA sample is incubated with a bisulfitesolution of a concentration range between 0.1 mol/l to 6 mol/l in thepresence of a denaturing reagent and/or solvent and at least onescavenger. In said patent application, several suitable denaturingreagents and scavengers are described. Desulfonation of the deaminatednucleic acid is afforded by incubation of the solution under alkalineconditions.

Patent application WO 03/038121 (US 20040115663) discloses a method inwhich the DNA to be analysed is bound to a solid surface during thebisulfite treatment. Consequently, purification and washing steps arefacilitated.

Patent application WO 04/067545 discloses a method in which the DNAsample is denatured by heat and incubated with a bisulfite solution of aconcentration range between 3 mol/l to 6.25 mol/l. Thereby the pH valueis between 5.0 and 6.0 and the nucleic acid is deaminated. Deaminatednucleic acids are desulfonated by incubation of the solution underalkaline conditions.

The art-recognized understanding that a ‘bisulfite conversion’ typicallycomprises a desulfonation step is illustrated in WO 04/067545:

-   -   “According to the invention the term a “bisulfite reaction”,        “bisulfite treatment” or “bisulfite method” shall mean a        reaction for the conversion of a cytosine base, preferably        cytosine bases, in a nucleic acid to an uracil base, preferably        uracil bases, in the presence of bisulfite ions whereby        preferably a 5-methyl-cytosine base, preferably        5-methyl-cytosine bases, is not significantly converted. This        reaction for the detection of methylated cytosines is described        in detail by Frommer et al., supra and Grigg and Clark        (Grigg, G. and Clark, S., Bioessays 16:431-436, 1994). The        bisulfite reaction contains a deamination step and a        desulfonation step, which can be conducted separately or        simultaneously (see FIG. 1; Grigg and Clark, supra). The        statement that 5-methyl-cytosine bases are not significantly        converted shall only take the fact into account that it cannot        be excluded that a small percentage of 5-methyl-cytosine bases        is converted to uracil although it is intended to convert only        and exclusively the (non-methylated) cytosine bases (Frommer et        al., supra). The expert skilled in the art knows how to perform        the bisulfite reaction, e.g. by referring to Frommer et al.,        supra or Grigg and Clark, supra who disclose the principal        parameters of the bisulfite reaction.”

Moreover, WO 04/067545 describes the general state of the art withregard to the different protocols:

-   -   “From Grunau et al., supra, it is known to the expert in the        field what variations of the bisulfite method are possible. In        summary, in the deamination step a buffer containing bisulfite        ions, optionally chaotropic agents and optionally further        reagents as an alcohol or stabilizers as hydroquinone are        employed and the pH is in the acidic range. The concentration of        bisulfite is between 0.1 and 6 M bisulfite, preferably between 1        M and 5.5 M, the concentration of the chaotropic agent is        between 1 and 8 M, whereby preferably guanidinium salts are        employed, the pH is in the acidic range, preferably between 4.5        and 6.5, the temperature is between 0° C. and 90° C., preferably        between room temperature (25° C.) and 90° C., and the reaction        time is between 30 min and 24 hours or 48 hours or even longer,        but preferably between 1 hour and 24 hours. The desulfonation        step is performed by adding an alkaline solution or buffer as        e.g. a solution only containing a hydroxide, e.g. sodium        hydroxide, or a solution containing ethanol, sodium chloride and        sodium hydroxide (e.g., 38% EtOH, 100 mM NaCl, 200 mM NaOH) and        incubating at room temperature or elevated temperatures for        several min, preferably between 5 min and 60 min.”

Desulfonation is, therefore, an inherent feature of all of thesemethods, and in any case a desulfonation takes place before the nucleicacids are used as templates for amplification reactions, in order toprovide an ideal template for the polymerase utilized in subsequentreactions.

Patent application WO 05/038051 describes improvements for theconversion of unmethylated cytosine to uracil by treatment with abisulfite reagent. According to this method the reaction is carried outin the presence of 10-35% by volume, preferentially in the presence of20-30% by volume of dioxane, one of its derivatives or a similaraliphatic cyclic ether. The bisulfite reaction can also be carried outin the presence of a n-alkylene glycol compound, particularly in thepresence of their dialkyl ethers, and especially in the presence ofdiethylene glycol dimethyl ether (DME). These compounds can be presentin a concentration of 1-35% by volume, preferentially of 5-25% byvolume. The bisulfite conversion is conducted at a temperature in therange of 0-80° C. and that the reaction temperature is increased for 2to 5 times to a range of 85-100° C. briefly during the course of theconversion (thermospike). It is further preferred that the temperatureincreases to 85-100° C., in particular to 90-98° C. during thetemperature increase of brief duration.

Subsequent to a bisulfite treatment, usually short, specific fragmentsof a known gene are amplified and either completely sequenced (Olek A,Walter J, The pre-implantation ontogeny of the H19 methylation imprint.Nat Genet. 3:275-6, 1997) or individual cytosine positions are detectedby a primer extension reaction (Gonzalgo M L and Jones P A., Rapidquantitation of methylation differences at specific sites usingmethylation-sensitive single nucleotide primer extension (Ms-SNuPE).Nucleic Acids Res. 25:2529-31, 1997; WO 95/00669) or by enzymaticdigestion (Xiong Z, Laird P W., COBRA: a sensitive and quantitative DNAmethylation assay. Nucleic Acids Res. 25: 2535-4, 1997).

Another technique to detect hypermethylation is the so-calledmethylation specific PCR (MSP) (Herman J G, Graff J R, Myohanen S,Nelkin B D and Baylin S B., Methylation-specific PCR: a novel PCR assayfor methylation status of CpG islands. Proc Natl Acad Sci U S A. 93:9821-6, 1996). The technique is based on the use of primers thatdifferentiate between a methylated and a non-methylated sequence ifapplied after bisulfite treatment of said DNA sequence. The primereither contains a guanine at the position corresponding to the cytosinein which case it will after bisulfite treatment only bind if theposition was methylated. Or the primer contains an adenine at thecorresponding cytosine position and therefore only binds to said DNAsequence after bisulfite treatment if the cytosine was unmethylated andhas hence been altered by the bisulfite treatment so that it hybridizesto adenine. With the use of these primers, amplicons can be producedspecifically depending on the methylation status of a certain cytosineand will as such indicate its methylation state.

Another technique is the detection of methylation via a labeled probe,such as used in the so called Taqman™ PCR, also known as MethyLight™(U.S. Pat. No. 6,331,393). With this technique it became feasible todetermine the methylation state of single or of several positionsdirectly during PCR, without having to analyze the PCR products in anadditional step.

Additionally, detection by hybridization has also been described (Oleket al., WO 99/28498).

The treatment with bisulfite (or similar chemical agents or enzymes)with the effect of altering the base pairing behaviour of one type ofcytosine specifically, either the methylated or the unmethylated,thereby introducing different hybridisation properties, makes thetreated DNA more applicable to the conventional methods of molecularbiology, especially the polymerase based amplification methods, such asthe PCR.

Base excision repair. Base excision repair occurs in vivo to repair DNAbase damage involving relatively minor disturbances in the helical DNAstructure, such as deaminated, oxidized, alkylated or absent bases.Numerous DNA glycosylases are known in the art, and function in vivoduring base excision repair to release damaged or modified bases bycleavage of the glycosidic bond that links such bases to the sugarphosphate backbone of DNA (Memisoglu, Samson, Mutation Res., 451:39-51,2000). All DNA glycosylases cleave glycosidic bonds but differ in theirbase substrate specificity and in their reaction mechanisms.

Carry-over contamination of amplification reactions (e.g., PCR);inadequacy of the prior art. One widely recognized application of suchglycosylases is decontamination in PCR applications. In any such PCRamplification, 2 to the 30 (2³⁰) or more copies of a single template aregenerated. This very large amount of DNA produced helps in thesubsequent analysis, like in DNA sequencing according to the Sangermethod, but it can also become a problem when this amount of DNA ishandled in an analytical laboratory. Even very small reaction volumes,when inadvertently not kept in a closed vial, can lead to contaminationof the whole work environment with a huge number of DNA copies. TheseDNA copies may be templates for a subsequent amplification experimentperformed, and the DNA analysed subsequently may not be the actualsample DNA, but contaminating DNA from a previous experiment. This mayalso lead to positive negative controls that should not contain any DNAand therefore no amplification should be observed.

In practice, this problem can be so persistent that whole laboratoriesmay move to a new location, because contamination of the workenvironment makes it impossible to still carry out meaningful PCR basedexperiments. In a clinical laboratory, however, the concern is also thatcontaminating DNA may cause false results when performing moleculardiagnostics. This would mean that actually contaminating DNA that stemsfrom a previous patient is analyzed, instead of the actual sample to beinvestigated.

Therefore, measures have been implemented to avoid contamination. Thisinvolves, for example, a PCR amplification and detection in one tube ina real time PCR experiment. In this case, it is not required that a PCRtube be opened. After use, the tube will be kept closed and discardedand therefore the danger of contamination leading to false results isgreatly reduced.

In addition, molecular means exist that reduce the risk ofcontamination. In a polymerase chain reaction, the enzymeuracil-DNA-glycosylase (UNG) reduces the potential for false positivereactions due to amplicon carryover (see e.g. U.S. Pat. No. 5,035,996 orThornton C G, Hartley J L, Rashtchian A., Utilizing uracil DNAglycosylase to control carryover contamination in PCR: characterizationof residual UDG activity following thermal cycling. Biotechniques,13:1804, 1992). The principle of this contamination protection method isthat in any amplification instead of dTTP dUTP is provided andincorporated and the resulting amplicon can be distinguished from itstemplate and any future sample DNA by uracil being present instead ofthymine. Prior to any subsequent amplification, uracil DNA-glycosylase(UNG) is used to cleave these bases from any contaminating DNA, andtherefore only the legitimate template remains intact and can beamplified. This method is considered the standard method of choice inthe art and is widely used in DNA based diagnostics. The following is acitation from a publication that summarizes the use of UNG (Longo M C,Berninger M S, Hartley J L., Use of uracil DNA glycosylase to controlcarry-over contamination in polymerase chain reactions. Gene., 93:125-8,1990):

-   -   “Polymerase chain reactions (PCRs) synthesize abundant        amplification products. Contamination of new PCRs with trace        amounts of these products, called carry-over contamination,        yields false positive results. Carry-over contamination from        some previous PCR can be a significant problem, due both to the        abundance of PCR products, and to the ideal structure of the        contaminant material for re-amplification. We report that        carry-over contamination can be controlled by the following two        steps: (i) incorporating dUTP in all PCR products (by        substituting dUTP for dTTP, or by incorporating uracil during        synthesis of the oligodeoxyribonucleotide primers; and (ii)        treating all subsequent fully preassembled starting reactions        with uracil DNA glycosylase (UNG), followed by thermal        inactivation of UNG. UNG cleaves the uracil base from the        phosphodiester backbone of uracil-containing DNA, but has no        effect on natural (i.e., thymine-containing) DNA. The resulting        apyrimidinic sites block replication by DNA polymerases, and are        very labile to acid/base hydrolysis. Because UNG does not react        with dUTP, and is also inactivated by heat denaturation prior to        the actual PCR, carry-over contamination of PCRs can be        controlled effectively if the contaminants contain uracils in        place of thymines.”

Another method for carry over protection in PCR has been described byWalder et al (Walder R Y, Hayes J R, Walder J A., Use of PCR primerscontaining a 3-terminal ribose residue to prevent cross-contamination ofamplified sequences. Nucleic Acids Res., 21:4339-43, 1993). Walder et aldescribe that carry over protection can be achieved—however not veryreproducibly—by using primers consisting of a 3′-end which ischaracterized as a ribo-cytidine. After primer extension theamplification product is cleaved specifically at the site of thisribonucleotide by an enzyme known as RNase A. That way the potentiallycontaminating amplificates are shortened at their ends and cannot servea templates for said primers in the following amplification procedure.However, a significant disadvantage inherent to this method is theinstability of the primer molecules, containing a ribonucleotide at the3′-end.

All of the documents cited herein are hereby incorporated by referencein its entirety.

Substantial problem in the prior art. Because the existence of uracilsis an inherent feature of bisulfite converted DNA and the necessaryproperty relied upon for detecting methylation differences, the priorart method of choice for carry over protection based onuracil-DNA-glycosylase enzyme activity, as described above, cannot beapplied. This limitation is very unfortunate, because a number ofpowerful assays for diagnosis are based on PCR performed on bisulfiteconverted DNA as a template. The difficulty of solving the problem fordecontamination of bisulfite converted templates is considered a generalone, that can not be solved by adaptation of the standard UNG method, asany bisulfite converted DNA will contain uracil as well. It hastherefore commonly been argued that, in any uracil-DNA-glycosylase step,the template DNA would be destroyed along with any contaminating DNA.

Therefore, there is a pronounced need in the art for new methods forcarry over prevention that have utility for routine performance of suchassays. There is a pronounced need in the art to provide solutions tothe problem of how to achieve a reliable carry over protection whenanalysing methylation of cytosine positions in DNA from patient samples.

SUMMARY OF ASPECTS OF THE INVENTION

Presently, no method has been reported to decontaminate DNA samples thatwould be compatible with bisulfite treated DNA employed as the templatefor an amplification procedure like PCR. In preferred aspects of thepresent invention, novel methods are provided to render the mostcommonly used method, based on use of the glycosylase enzyme UNG asdescribed above, applicable to DNA methylation analysis.

Surprisingly, the instant inventors were able to solve the problem ofproviding reliable carry-over protection in the context of methylationassays. Preferred aspects comprise sulfonating, or sulfonating anddeaminating unmethylated cytosines, without a subsequent desulfonation.After the unmethylated cytosines are converted to C6-sulfonated uracils,the reaction mixture is treated with uracil-DNA-glycosylase (UNG), whichdegrades all nonsulfonated-uracil-containing DNA and hence everycontaminating DNA, but has no effect on the sulfonated-uracil-containingDNA. In particular aspects, a deactivation of the UNG followed by adesulfonation of the sulfonated uracils is carried out.

The discovery, reported upon for the first time in aspects of thisapplication, that sulfonation of uracil at the C6 position protects theuracil from being degraded by UNG, provides a new and surprisingsolution to the above stated problem. Particular embodiments of thepresent invention, therefore, comprise methods that provides both asufficient and reliable differentiation between methylated andunmethylated cytosines, as well as the applicability, in the context ofmethylation analysis, of the gold standard of carry over protection(based on use of UNG) for common PCR based assays.

Particular aspects disclose methods for the specific amplification oftemplate DNA in the presence of potentially contaminating PCR productsfrom previous amplification experiments. This template DNA is usuallyderived from isolating the genomic DNA to be analyzed before the methodcan be applied. Also, the template nucleic acid used in this method isusually already denatured and therefore present in a single strandedmodus. In a first step of this representative embodiment, DNA iscontacted with a bisulfite solution, which reacts with unmethylatedcytosines but not with methylated cytosines, by sulfonating them. Thisresults in a modification of said nucleic acids, which is known assulfonation. This sulfonation of unmethylated cytosine in aqueoussolution results in deamination of the cytosine whereby sulfonateduracil is generated.

It has herein, for the first time, been recognized that such sulfonation(which occurs only at the unmethylated cytosine bases) protects thetemplate nucleic acid from being a target for the enzyme UNG, andthereby allows for discrimination of template nucleic acid andpotentially contaminating nucleic acids. Any contaminating DNA, whichcontains unprotected unsulfonated or desulfonated uracils while UNG isactive, is subsequently degraded enzymatically and only the templatenucleic acid from the sample remains to be amplified in the next step.

After treatment with UNG has been accomplished and UNG activity isterminated, the sulfonated uracil bases, which replace the unmethylatedcytosines, are converted into uracil by desulfonation. The method isuseful for decontamination of nucleic acid samples, or for example, foravoiding amplification of ‘carry over products,’ and in particular inthe context of DNA methylation analysis.

Particular aspects provide a method for providing a decontaminatedtemplate nucleic acid for polymerase based amplification reactionssuitable for DNA methylation analysis, comprising: incubating nucleicacids with a bisulfite reagent solution, whereby the unmethylatedcytosines within said nucleic acid are sulfonated, or sulfonated anddeaminated, and mixing the sulfonated or sulfonated and deaminatedtemplate nucleic acid with the components required for a polymerasemediated amplification reaction or an amplification based detectionassay; adding to this mixture UNG and incubating the mixture, wherebynucleic acids containing non-sulfonated uracil are degraded, andterminating UNG activity and desulfonating the template nucleic acid,thereby converting unmethylated deaminated and sulfonated cytosines,i.e. sulfonated uracils into uracils.

In additional embodiments, a polymerase based amplification oramplification based assay is subsequently performed, which preferablytakes place in the presence of dUTPs instead of dTTPs.

Preferably, the polymerase activity is started during desulfonationstep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes the complete conversion of unmethylated cytosine touracil, so called bisulfite conversion. The first step of this reactiontakes place when unmethylated cytosine bases are contacted withhydrogensulfite at a ph around 5. The sulfonation takes place atposition 6 of the cyclic molecule (C6 position). The second step is thedeamination that takes place rather spontaneously in aqueous solutionand thereby converts cytosine sulfonate into uracil sulfonate. The thirdstep is the desulfonation step, which takes place in alkalineconditions, resulting in uracil.

FIG. 2 shows a plot of real time amplification of methylated DNA of theGSTP1 gene from desulfonated bisulfite converted DNA, according to thestate of the art. The Y-axis shows the fluorescence signal measured inchannel F2 normalized against channel F1 at each cycle (X-axis). 10 ng,1 ng respective 0.1 ng bisulfite treated methylated DNA were added tothe reaction. No signals were determined using the reaction mixcontaining Uracil-DNA-glycosylase (labeled in open circles) indicating acomplete degradation of the bisulfite converted DNA. Amplificationoccurs only in absence of Uracil-DNA-glycosylase (labeled inrectangles). No template control is marked as solid line.

FIG. 3 is a plot of real time amplification of methylated DNA of theGSTP1 gene from bisulfite converted DNA according to the claimed newmethod without desulfonation. The Y-axis shows the fluorescence signalmeasured in channel F2 normalized against channel F1 at each cycle(X-axis). 10 ng, 1 ng respective 0.1 ng bisulfite treated methylated DNAwere added to the reaction. The signals generated from reaction withoutUracil-DNA-glycosylase are labeled with circles. No significantdifference in amplification was determined from the reaction containingUracil-DNA-glycosylases (labeled in triangles) indicating that6-Sulfon-Uracil containing DNA is not a template for UNG. No templatecontrol is marked as solid line.

FIG. 4 demonstrates of the efficient degradation of uracil containingDNA. The plot shows the reamplification of PCR products containinguracil. The Y-axis shows the fluorescence signal measured in channel F2normalized against channel F1 at each cycle (X-axis); 10⁵ copies wereadded to the reaction. The signals determined from reaction withoutUracil-DNA-glycosylase are labeled diamonds showing a high efficientreamplification. The reaction containing Uracil-DNA-glycosylases resultsin dramatically increased crossing points (labeled in stars) indicatinga strong degradation of uracil containing PCR products by UNG. Notemplate control is marked as solid line.

FIG. 5 shows a correlation plot of the results obtained in Example 3 bythe standard workflow and the method according to the invention (carryover prevention). Every symbol represents a single sample: quadratestumor tissues, triangles normal adjacent tissues. The percentage ofmethylation determined according to the standard workflow (x-axis) or tothe method according to the invention (y-axis) is indicated for eachsample. The exemplary inventive methods have led only in 2 out of 24samples to a different methylation percentage as the standard workflow.This means that although the samples treated according to the method ofthe invention were contaminated with uracil containing TPEF amplicons,only DNA of the samples served as a template for amplification of theTPEF amplicon in nearly all cases. In case of the said two samples, thediffering results occurred presumable because of the low methylationpercentage of the DNA (smaller than 0.2%).

DETAILED DESCRIPTION

Presently, no method has been reported to decontaminate DNA samples thatwould be compatible with bisulfite treated DNA employed as the templatefor an amplification procedure like PCR. In preferred aspects of thepresent invention, novel methods are provided to render the mostcommonly used method, based on use of the glycosylase enzyme UNG asdescribed above, applicable to DNA methylation analysis.

Particular embodiments provide for a method comprising the followingsteps:

first, incubating a template nucleic acids with a bisulfite reagentcontaining solution, whereby the unmethylated cytosines within saidnucleic acid are sulfonated, or sulfonated and deaminated;

second, mixing the sulfonated, or sulfonated and deaminated, templatenucleic acid with the components required for a polymerase mediatedamplification reaction or an amplification based detection assay;

third, adding to this mixture UNG units and incubating said mixture,whereby any nucleic acids containing non-sulfonated uracils aredegraded, whereas sulfonated uracils essentially remain intact; and

fourth, terminating the UNG activity, and desulfonating the templatenucleic acid.

The sulfonation takes place at C6 position of the base cytosine (FIG.1). Deamination of sulfonated cytosines takes place spontaneously inaqueous solution, and thereby the sulfonated cytosine is converted intoa sulfonated uracil.

Particular aspects are based on two essential discoveries. Firstly,applicants determined that sulfonated nucleic acids are stable up to atleast 6 days at 4° C.; e.g., when stored in a common laboratory fridge.This discovery was essential to the method according to the inventionbecause a spontaneous uncontrolled desulfonation of the nucleic acidswould render the method unreliable and unstable. Whereas, discoveringand knowing that the nucleic acid's sulfonation pattern, basicallyresembling the nucleic acids methylation pattern, will remain stablewhen stored for several days at a lower temperature allows the use ofthis feature in performing sensitive assays to detect exactly whichnucleic acids were methylated within a given sample to which extent.

Secondly, it was discovered by applicants that uracil-DNA-glycosylase(UNG) does not degrade nucleic acids containing sulfonated uracils; inother words sulfonated uracils are not a substrate for UNG activity, andtherefore are protected from degradation by UNG.

It was also determined by applicants that “real time PCR” assays usingsulfonated nucleic acids as template performed well in the presence ofUNG activity, indicating that sulfonated nucleic acids as derived fromthe first step of the bisulfite treatment can serve as templates in PCRbased assays.

Additionally, applicants determined that the desulfonation reaction,which must take place before the nucleic acid can be amplified by apolymerase mediated amplification, can be performed within the PCRreaction.

The developed methods, according to particular exemplary aspects of theinvention, were successfully tested for GSTP1 and connexine (seeExamples herein).

In the first step, the bisulfite mediated cytosine sulfonation may beinitiated according to the first steps of common bisulfite conversionprotocols as indicated above, in particular as indicated in WO 05/038051(incorporated by reference herein). The reaction may take place both insolution as well as also on DNA bound to a solid phase. Preferablysodium disulfite (=sodium bisulfite/sodium metabisulfite) is used, sinceit is more soluble in water than sodium sulfite. The disulfite saltdisproportionates in aqueous solution to the hydrogen sulfite anionsnecessary for the cytosine sulfonation. When bisulfite concentration isdiscussed in more detail, this refers to the concentration of hydrogensulfite and sulfite anions in the reaction solution. For the methodaccording to the invention, concentration ranges of 0.1 to 6 mol/l arepossible. Particularly preferred is a concentration range of 1 to 6mol/l, and most particularly preferred, 2-4 mol/l. However, when dioxaneis used as a denaturing agent, the maximal working concentration ofbisulfite is smaller. Dioxane may also be utilized in differentconcentrations. Preferably, the dioxane concentration amounts to 10 to35%, particularly preferred is 20 to 30%, and most particularlypreferred is 22 to 28%, especially 25%.

In particularly preferred embodiments with a dioxane concentration of22-28%, the final preferred bisulfite concentration amounts to 3.3 to3.6 mol/l, and in the most particularly preferred embodiment with adioxane concentration of 25%, it amounts to 3.5 mol/l (see Examples).

In another preferred embodiment, DME is used as denaturing agent indifferent concentrations. DME is used in concentrations in the range of1-35%, preferable in the range of 5-25%, and most preferably 10%.

In a particularly preferred embodiment the bisulfite conversion iscarried out in the presence of scavengers. The preferred scavengers arechromane derivatives, e.g., 6-hydroxy-2,5,7,8,-tetramethylchromane2-carboxylic acid (also known as: Trolox-C™). Further scavengers arelisted in the patent application WO 01/98528 (=DE 100 29 915; =U.S.application Ser. No. 10/311,661; incorporated herein in its entirety).

The bisulfite conversion can be conducted in a wide temperature rangefrom 0 to 95° C. However, in a preferred embodiment the reactiontemperature lies between 30-70° C. Particularly preferred is a rangebetween 45-60° C.; most particularly preferred between 50-55° C. Theoptimal reaction time of the bisulfite treatment depends on the reactiontemperature. The reaction time normally amounts to between 1 and 18hours (see: Grunau et al. 2001, Nucleic Acids Research; 29(13):E65-5.).The preferred reaction time is 4-6 hours for a reaction temperature of50° C.

In a particularly preferred embodiment of the method according to theinvention, the bisulfite conversion is conducted at mild reactiontemperatures, wherein the reaction temperature is then clearly increasedfor a short time at least once during the course of the conversion. Thetemperature increases of short duration are named “thermospikes” below.The “standard” reaction temperature outside the thermospikes is denotedas the basic reaction temperature. The basic reaction temperatureamounts to between 0 and 80° C., preferably between 30-70° C., mostpreferably 45°-55° C., as described above. The reaction temperatureduring a thermospike is increased to over 85° C. by at least onethermospike. The optimal number of thermospikes is a function of thebasic reaction temperature. The higher the optimal number ofthermospikes is, the lower is the basic reaction temperature. In suchembodiments, at least one thermospike is necessary in each case. And, onthe other hand, in principle, any number of thermospikes is conceivable.

In a particular embodiment, the preferred number of thermospikes isbetween 1 and 10 thermospikes, depending on the basic reactiontemperature. Two to five thermospikes are particularly preferred. Duringthe thermospikes the reaction temperature increases preferably to 85 to100° C., particularly preferably to 90-98° C., and most preferably to94° C.-96° C. The duration in time of the temperature increases alsodepends on the volume of the reaction batch. The duration in time of thethermospikes also depends on the volume of the reaction batch. It mustbe assured that the temperature is increased uniformly throughout thetotal reaction solution. For a 20 μl reaction batch when using athermocycler a duration between 15 seconds and 1.5 minutes, especially aduration between 20 and 50 seconds is preferred. In a particularpreferred embodiment the duration is 30 seconds. Operating on a volumeof 100 μl the preferred range lies between 30 seconds and 5 minutes,especially between 1 and 3 minutes. Particularly preferred are 1.5minutes. For a volume of 600 μl, a duration of 1 to 6 minutes ispreferred, especially between 2 and 4 minutes. Particularly preferred isa duration of 3 minutes. A person skilled in the art will easily be ableto determine suitable durations of thermospikes in relation to a varietyof reaction volumes.

The above described use of thermospikes leads to a significantly betterconversion rates in the bisulfite conversion reaction, even when theabove-described denaturing solvents are not utilized. According toadditional aspects of the invention, a method for bisulfite conversionof DNA is hereby characterized in that the basic reaction temperatureamounts to 0° C. to 80° C. and that the reaction temperature isincreased for a short time to over 85° C. at least once in the course ofthe conversion.

In the second step, prior to any desulfonation step, units of an enzymeactivity, which specifically degrades non-sulfonated uracil containingnucleic acids, are added to said premix. In a preferred embodiment, thisdegrading enzyme is a DNA-glycosylase or an endonuclease, inparticularly UNG (uracil-DNA-glycosylase). The contaminating nucleicacid is characterized, for example, in that it contains non-sulfonateduracil bases. The added degrading enzyme is characterized by cleavingthe non-sulfonated uracil base from the phosphodiester backbone ofnon-sulfonated uracil-containing nucleic acids, but has no effect onsulfonated-uracil containing nucleic acid or on thymine containingnucleic acid, that does not contain uracil. The resulting apyrimidinicsites block replication by DNA polymerases, and are very labile toacid/base hydrolysis.

In another preferred embodiment, the first step is carried out asdescribed above. Thereafter, in an intermediate step, the sulfonatedand/or deaminated nucleic acid is mixed with components required for apolymerase mediated amplification reaction or an amplification baseddetection assay. The amplification reaction mix is prepared according tostandard protocols. Such an amplification mix, preferably a PCR mix, maycontain at least one primer set of two primers and a polymerase. Thispolymerase preferably is a heat stable enzyme, even more preferred isthe use of a thermally activated polymerase for hot start PCR, and mostparticularly preferred a thermally activated Taq polymerase is used.

The following second step is also carried out as described above. Unitsof an enzyme activity, which specifically degrades sulfonated-uracilcontaining nucleic acid, are added to said premix. The sulfonated samplenucleic acid and a set of at least two primer oligonucleotides areincubated with a composition of enzymes, including an enzyme withsulfonated-uracil containing nucleic acid degrading activity and buffersto cleave or degrade any contaminating nucleic acid. The contaminatingnucleic acid is characterized in that it contains uracil bases. Theadded degrading enzyme activity is characterized by cleaving the uracilbase from the phosphodiester backbone of non-sulfonated uracilcontaining nucleic acid, but has no effect on sulfonated uracilcontaining nucleic acid or on thymine containing nucleic acid, that doesnot contain uracil. The resulting apyrimidinic sites block replicationby DNA polymerases, and are very labile to acid/base hydrolysis. Inprinciple, the enzymatic activity is any enzymatic activity, whichcauses specifically apyrimidinic sites or one or more nicks adjacent toa non-sulfonated uracil base. In any case this will result in a block ofthe replication by DNA polymerase.

The primer oligonucleotides will be chosen such that they amplify afragment of interest. It is particularly preferred that these primersare designed to amplify a nucleic acid fragment of a template nucleicacid sample by means of a polymerase reaction, in particular apolymerase chain reaction, as known in the art. The primeroligonucleotides are therefore designed to anneal to the templatenucleic acids to form a double strand, following the Watson-Crick basepairing rules, and the length of these oligonucleotide primers will beselected such that they anneal at approximately the same temperature.

In said second step, an enzyme and the matching buffers are added toachieve cleavage of any present, contaminating amplificates that weregenerated in any of the preceding experiments. hese amplificates willhave the property that they comprise uracil bases instead of thyminebases, if generated in a polymerase reaction providing dUTPs instead ofdTTPs. Therefore, not the sample nucleic acid at this step would berecognized and degraded by the enzyme, but only nucleic acids that weregenerated in preceding amplifications, the contaminating DNA that has tobe removed before the next round of amplification.

It is particularly preferred that the enzyme employed in this secondstep is uracil-DNA-glycosylase (UNG). It is further preferred that saidnon-sulfonated uracil containing nucleic acid degrading enzyme isthermolabile, in particular the DNA-glycosylase or the Endonuclease arethermolabile, respectively, and most particularly preferred the UNG isthermolabile.

In the third step, after enzymatic degradation, the composition ofenzymes and buffer is subsequently inactivated, in that it is notcapable of substantially cleaving any product of the subsequentamplification step. The nonsulfonated-uracil-containing nucleic aciddegrading enzyme activity is terminated, in particular theDNA-glycosylase activity or the endonuclease activity is terminated, andmost particularly preferred the UNG activity is terminated.

After the composition of non-sulfonated uracil containing nucleic aciddegrading enzymes and buffer is inactivated, in that it is not capableof substantially degrading any product of the subsequent amplificationstep, the fourth step is performed; that is, the desulfonation of thetemplate nucleic acids. Prior to the amplification by a polymerase, afourth step must be conducted, that is the desulfonation of thesulfonated template nucleic. Desulfonation may take place under alkalineconditions (as described in the art). Desulfonation however may also becatalyzed by an increase in temperature under pH conditions as they arecommon to the PCR reaction.

It is a preferred embodiment of the invention that steps 3 and 4 areconducted simultaneously by a short increase of the incubationtemperature of said premix, which results in deactivation of thenon-sulfonated uracil containing nucleic acid degrading enzyme on theone hand and in thermal desulfonation of the template nucleic acid, onthe other hand. This increase in the incubation temperature can also besuitable to transfer double-stranded DNA into single-stranded formenabling an amplification.

The sample nucleic acid may now be amplified in the next step using theset of primer oligonucleotides and a polymerase, while any cleavedcontaminating DNA is essentially not amplified. The sample nucleic acidmay be amplified, using a set of primer oligonucleotides and apolymerase, while the cleaved or degraded contaminating nucleic acidcannot be amplified. The amplified products may now be analyzed and themethylation status in the genomic DNA may be deduced from the presenceof an amplified product and/or from the analysis of the sequence withinthe amplified product.

This amplification may be carried out, in a particularly preferredembodiment of the invention, by means of a polymerase chain reaction,but also by other means of DNA amplification known in the art, like TMA(transcription mediated amplification), isothermal amplifications,rolling circle amplification, ligase chain reaction, and others.

In particular embodiments, the generated DNA fragments will then beanalyzed, concerning their presence, the amount, or their sequenceproperties or a combination thereof.

Therefore one embodiment of the invention is a method for providing adecontaminated template nucleic acid for polymerase based amplificationreactions suitable for DNA methylation analysis, which is characterizedby: firstly, incubating a template nucleic acid with a bisulfite reagentcontaining solution, whereby the unmethylated cytosines within saidnucleic acid are sulfonated and/or deaminated; secondly, mixing thesulfonated and/or deaminated template nucleic acid with the componentsrequired for a polymerase mediated amplification reaction or anamplification based detection assay; thirdly, adding to this mixture anenzyme with uracil-DNA-glycosylase activity and incubating the mixture,whereby nucleic acids containing non-sulfonated uracils are degraded;fourthly, terminating the UNG activity; and fifthly, desulfonating thetemplate nucleic acid.

In a preferred embodiment, the method is further characterized by a step4 and 5 taking place simultaneously, by briefly incubating the mixtureat an increased temperature, whereby the UNG activity is terminated,whereby desulfonation of the template nucleic acid takes place, andwhereby the DNA is transferred from a double-stranded form into asingle-stranded form suitable for amplification.

In a further preferred embodiment, the template nucleic acid isamplified in a subsequent step 6.

It is further preferred that upon termination of the non-sulfonateduracil containing nucleic acid degrading activity and desulfonation ofthe template nucleic acid, a polymerase based amplification reaction isstarted and/or an amplification based assay is performed.

It is further preferred that the polymerase based amplification reactionis started by a brief incubation at increased temperature (heatactivation).

In a preferred embodiment of the method the polymerase is a heat stablepolymerase.

It is particularly preferred that the polymerase mediated amplificationor amplification based assay is performed in the presence of dUTPsinstead of dTTPs.

In one preferred embodiment, the method is performed by adding an amountof units of the enzyme, which specifically degradesnonsulfonated-uracil-containing nucleic acids; in the second step thatis required to degrade essentially all potential contaminating nucleicacids.

It is especially preferred that upon activation of the polymerase enzymea polymerase based amplification reaction or an amplification basedassay is performed.

It is further preferred that upon activation of the polymerase enzyme apolymerase based amplification reaction or amplification based assay isperformed in the presence of dUTPs instead of dTTPs.

It is further preferred that this assay is a real time assay.

In a particularly preferred embodiment, the sample DNA is obtained fromserum or other body fluids of an individual. Preferably, the DNA samplesare obtained from cell lines, tissue embedded in paraffin, for exampletissue from eyes, intestine, kidneys, brain, heart, prostate, lungs,breast or liver, histological slides, body fluids and all possiblecombinations thereof. The term body fluids is meant to comprise fluidssuch as whole blood, blood plasma, blood serum, urine, sputum,ejaculate, semen, tears, sweat, saliva, lymph fluid, bronchial lavage,pleural effusion, peritoneal fluid, meningal fluid, amniotic fluid,glandular fluid, fine needle aspirates, nipple aspirate fluid, spinalfluid, conjunctival fluid, vaginal fluid, duodenal juice, pancreaticjuice, bile, stool and cerebrospinal fluid. It is especially preferredthat said body fluids are whole blood, blood plasma, blood serum, urine,stool, ejaculate, bronchial lavage, vaginal fluid and nipple aspiratefluid.

In a particularly preferred embodiment, the chemical treatment isconducted with a bisulfite (=disulfite, hydrogen sulfite). It is againpreferred that the chemical treatment is conducted after embedding theDNA in agarose, or that it is conducted in the presence of a denaturingagent and/or a radical scavenger.

The following methylation detection assays are all preferred embodimentsof the invention when performed subsequently to the steps of the methodaccording to the invention:

Methylation Assay Procedures. Various methylation assay procedures areknown in the art, and can be used in conjunction with the presentinvention. These assays allow for determination of the methylation stateof one or a plurality of CpG dinucleotides (e.g., CpG islands) within aDNA sequence. Such assays involve, among other techniques, DNAsequencing of bisulfite-treated DNA, and a number of PCR basedmethylation assays, some of them—known as COBRA, MS-SNuPE, MSP, nestedMSP, HeavyMethyl and MethyLight—are described in more detail now.

Bisulfite Sequencing. DNA methylation patterns and 5-methylcytosinedistribution can be analyzed by sequencing analysis of a previouslyamplified fragment of the bisulfite treated genomic DNA, as described byFrommer et al. (Frommer et al. Proc. Natl. Acad. Sci. USA 89:1827-1831,1992). As the bisulfite treated DNA is amplified before sequencing, theamplification procedure according to the invention may be used incombination with this detection method.

Cobra. COBRA analysis is a quantitative methylation assay useful fordetermining DNA methylation levels at specific gene loci in smallamounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534,1997). Briefly, restriction enzyme digestion is used to revealmethylation-dependent sequence differences in PCR products of sodiumbisulfite-treated DNA. Methylation-dependent sequence differences arefirst introduced into the genomic DNA by standard bisulfite treatmentaccording to the procedure described by Frommer et al. (Proc. Natl.Acad. Sci. USA 89:1827-1831, 1992) or as described by Olek et al (OlekA, Oswald J, Walter J. (1996) Nucleic Acids Res. 24: 5064-6). PCRamplification of the bisulfite converted DNA is then performed usingmethylation unspecific primers followed by restriction endonucleasedigestion, gel electrophoresis, and detection using specific, labeledhybridization probes. Methylation levels in the original DNA sample arerepresented by the relative amounts of digested and undigested PCRproduct in a linearly quantitative fashion across a wide spectrum of DNAmethylation levels. In addition, this technique can be reliably appliedto DNA obtained from microdissected paraffin-embedded tissue samples.Typical reagents (e.g., as might be found in a typical COBRA-based kit)for COBRA analysis may include, but are not limited to: PCR primers forspecific gene (or methylation-altered DNA sequence or CpG island);restriction enzyme and appropriate buffer; gene-hybridization oligo;control hybridization oligo; kinase labeling kit for oligo probe; andradioactive nucleotides. Additionally, bisulfite conversion reagents mayinclude: DNA denaturation buffer; sulfonation buffer; DNA recoveryreagents or kits (e.g., precipitation, ultrafiltration, affinitycolumn); desulfonation buffer; and DNA recovery components.Additionally, restriction enzyme digestion of PCR products amplifiedfrom bisulfite-converted DNA is also used, in the method described bySadri & Homsby (Nucl. Acids Res. 24:5058-5059, 1996). The bisulfiteconversion and amplification procedure according to the invention may beused in combination with this detection method.

Ms-SNuPE (Methylation-Sensitive Single Nucleotide Primer Extension).

The Ms-SNuPE technique is a quantitative method for assessingmethylation differences at specific CpG sites based on bisulfitetreatment of DNA, followed by single-nucleotide primer extension(Gonzalgo & Jones, Nucleic Acids Res. 25:2529-2531, 1997). Briefly,genomic DNA is reacted with sodium bisulfite to convert unmethylatedcytosine to uracil while leaving 5-methylcytosine unchanged.Amplification of the desired target sequence is then performed using PCRprimers specific for bisulfite-converted DNA, and the resulting productis isolated and used as a template for methylation analysis at the CpGsite(s) of interest. Small amounts of DNA can be analyzed (e.g.,microdissected pathology sections), and it avoids utilization ofrestriction enzymes for determining the methylation status at CpG sites.Typical reagents (e.g., as might be found in a typical Ms-SNuPE-basedkit) for Ms-SNuPE analysis may include, but are not limited to: PCRprimers for specific gene (or methylation-altered DNA sequence or CpGisland); optimized PCR buffers and deoxynucleotides; gel extraction kit;positive control primers; Ms-SNuPE primers for specific gene; reactionbuffer (for the Ms-SNuPE reaction); and radioactive nucleotides.Additionally, bisulfite conversion reagents may include: DNAdenaturation buffer; sulfonation buffer; DNA recovery regents or kit(e.g., precipitation, ultrafiltration, affinity column); desulfonationbuffer; and DNA recovery components. The bisulfite conversion andamplification procedure according to the invention may be used incombination with this detection method.

MSP. MSP (methylation-specific PCR) allows for assessing the methylationstatus of virtually any group of CpG sites within a CpG island,independent of the use of methylation-sensitive restriction enzymes(Herman et al. Proc. Natl. Acad. Sci. USA 93:9821-9826, 1996; U.S. Pat.No. 5,786,146). Briefly, DNA is modified by sodium bisulfite convertingall unmethylated, but not methylated cytosines to uracil, andsubsequently amplified with primers specific for methylated versusunmethylated DNA. MSP primer pairs contain at least one primer, whichhybridizes to a bisulfite treated CpG dinucleotide. Therefore, thesequence of said primers comprises at least one CpG dinucleotide. MSPprimers specific for non-methylated DNA contain a ‘T’ at the 3′ positionof the C position in the CpG. Preferably, therefore, the base sequenceof said primers is required to comprise a sequence having a length of atleast 9 nucleotides which hybridizes to the bisulfite converted nucleicacid sequence, wherein the base sequence of said oligomers comprises atleast one CpG dinucleotide. MSP requires only small quantities of DNA,is sensitive to 0.1% methylated alleles of a given CpG island locus, andcan be performed on DNA extracted from paraffin-embedded samples.Typical reagents (e.g., as might be found in a typical MSP-based kit)for MSP analysis may include, but are not limited to: methylated andunmethylated PCR primers for specific gene (or methylation-altered DNAsequence or CpG island), optimized PCR buffers and deoxynucleotides, andspecific probes. The bisulfite conversion and amplification procedureaccording to the invention may be used in combination with thisdetection method.

Nested MSP (Belinsky and Palmisano in US application 20040038245).Considering the apparent conflict of requiring high specificity of theMSP primer to sufficiently differentiate between CG and TG positions butallowing for a mismatch in order to create a unique restriction site itis preferred to use an amended version of MSP, known as nested MSP, asdescribed in WO 02/18649 and US patent application 20040038245 byBelinsky and Palmisano. This method to detect the presence ofgene-specific promoter methylation, comprises the steps of: expandingthe number of copies of the genetic region of interest by using apolymerase chain reaction to amplify a portion of said region where thepromoter methylation resides, thereby generating an amplificationproduct; and using an aliquot of the amplification product generated bythe first polymerase chain reaction in a second, methylation-specific,polymerase chain reaction to detect the presence of methylation. Inother words a non methylation specific PCR is performed prior to themethylation specific PCR. The bisulfite conversion and amplificationprocedure according to the invention may be used in combination withthis detection method.

Heavymethyl™. (WO 02/072880; Cottrell SE et al. Nucleic Acids Res. 2004Jan 13;32(1):e10) A further preferred embodiment of the method comprisesthe use of blocker oligonucleotides. In the HeavyMethyl™ assay blockingprobe oligonucleotides are hybridized to the bisulfite treated nucleicacid concurrently with the PCR primers. PCR amplification of the nucleicacid is terminated at the 5′ position of the blocking probe, such thatamplification of a nucleic acid is suppressed where the complementarysequence to the blocking probe is present. The probes may be designed tohybridize to the bisulfite treated nucleic acid in a methylation statusspecific manner. For example, for detection of methylated nucleic acidswithin a population of unmethylated nucleic acids, suppression of theamplification of nucleic acids which are unmethylated at the position inquestion would be carried out by the use of blocking probes comprising a‘CpA’ or ‘TpA’ at the position in question, as opposed to a ‘CpG’ if thesuppression of amplification of methylated nucleic acids is desired. ForPCR methods using blocker oligonucleotides, efficient disruption ofpolymerase-mediated amplification requires that blocker oligonucleotidesnot be elongated by the polymerase. Preferably, this is achieved throughthe use of blockers that are 3′-deoxyoligonucleotides, oroligonucleotides derivatized at the 3′ position with other than a “free”hydroxyl group. For example, 3′—O—acetyl oligonucleotides arerepresentative of a preferred class of blocker molecule. Additionally,polymerase-mediated decomposition of the blocker oligonucleotides shouldbe precluded. Preferably, such preclusion comprises either use of apolymerase lacking 5′-3′ exonuclease activity, or use of modifiedblocker oligonucleotides having, for example, thioate bridges at the5′-terminii thereof that render the blocker molecule nuclease-resistant.Particular applications may not require such 5′ modifications of theblocker. For example, if the blocker- and primer-binding sites overlap,thereby precluding binding of the primer (e.g., with excess blocker),degradation of the blocker oligonucleotide will be substantiallyprecluded. This is because the polymerase will not extend the primertoward, and through (in the 5′-3′ direction) the blocker—a process thatnormally results in degradation of the hybridized blockeroligonucleotide.

A particularly preferred blocker/PCR embodiment, for purposes of thepresent invention and as implemented herein, comprises the use ofpeptide nucleic acid (PNA) oligomers as blocking oligonucleotides. SuchPNA blocker oligomers are ideally suited, because they are neitherdecomposed nor extended by the polymerase. Preferably, therefore, thebase sequence of said blocking oligonucleotide is required to comprise asequence having a length of at least 9 nucleotides which hybridizes tothe chemically treated nucleic acid sequence, wherein the base sequenceof said oligonucleotides comprises at least one CpG, TpG or CpAdinucleotide. The bisulfite conversion and amplification procedureaccording to the invention may be used in combination with thisdetection method.

Preferably, real-time PCR assays are performed specified by the use ofsuch primers according to the invention. Real-time PCR assays can beperformed with methylation specific primers (MSP-real time) asmethylation-specific PCR (“MSP”; as described above), or withnon-methylation specific primers in presence of methylation specificblockers (HM real-time) (“HEAVYMETHYL”, as described above). Real-timePCR may be performed with any suitable detectably labelledlabeledprobes. For details see below.

Both of these methods (MSP or HM) can be combined with the detectionmethod known as MethyLight™ (a fluorescence-based real-time PCRtechnique) (Eads et al., Cancer Res. 59:2302-2306, 1999), whichgenerally increases the specificity of the signal generated in such anassay. Whenever the real-time probe used is methylation specific initself, the technology shall be referred to as MethyLight™, a widelyused method.

Another assay makes use of the methylation specific probe, the so called“QM” (quantitative methylation) assay. A methylation unspecific,therefore unbiased real-time PCR amplification is performed which isaccompanied by the use of two methylation specific probes (MethyLight™)one for the methylated and a second for the unmethylated amplificate.That way two signals are generated which can be used to a) determine theratio of methylated (CG) to unmethylated (TG) nucleic acids, and at thesame time b) the absolute amount of methylated nucleic acids can bedetermined, when calibrating the assay with a known amount of controlDNA.

MethyLight™.

The MethyLight™ assay is a high-throughput quantitative methylationassay that utilizes fluorescence-based real-time PCR (TaqMan™)technology that requires no further manipulations after the PCR step(Eads et al., Cancer Res. 59:2302-2306, 1999). Briefly, the MethyLight™process begins with a mixed sample of genomic DNA that is converted, ina sodium bisulfite reaction, to a mixed pool of methylation-dependentsequence differences according to standard procedures (the bisulfiteprocess converts unmethylated cytosine residues to uracil).Fluorescence-based PCR is then performed either in an “unbiased” (withprimers that do not overlap known CpG methylation sites) PCR reaction,or in a “biased” (with PCR primers that overlap known CpG dinucleotides)reaction. Sequence discrimination can occur either at the level of theamplification process or at the level of the fluorescence detectionprocess, or both.

The MethyLight™ assay may be used as a quantitative test for methylationpatterns in the genomic DNA sample, wherein sequence discriminationoccurs at the level of probe hybridization. In this quantitativeversion, the PCR reaction provides for unbiased amplification in thepresence of a fluorescent probe that overlaps a particular putativemethylation site. An unbiased control for the amount of input DNA isprovided by a reaction in which neither the primers, nor the probeoverlie any CpG dinucleotides. Alternatively, a qualitative test forgenomic methylation is achieved by probing of the biased PCR pool witheither control oligonucleotides that do not “cover” known methylationsites (a fluorescence-based version of the “MSP” technique), or witholigonucleotides covering potential methylation sites.

The MethyLight™ process can by used with a “TaqMan®” probe in theamplification process. For example, double-stranded genomic DNA istreated with sodium bisulfite and subjected to one of two sets of PCRreactions using TaqMan® probes; e.g., with either biased primers andTaqMan® probe, or unbiased primers and TaqMan® probe. The TaqMan® probeis dual-labeled with fluorescent “reporter” and “quencher” molecules,and is designed to be specific for a relatively high GC content regionso that it melts out at about 10° C. higher temperature in the PCR cyclethan the forward or reverse primers. This allows the TaqMan® probe toremain fully hybridized during the PCR annealing/extension step. As theTaq polymerase enzymatically synthesizes a new strand during PCR, itwill eventually reach the annealed TaqMan® probe. The Taq polymerase 5′to 3′ endonuclease activity will then displace the TaqMan®) probe bydigesting it to release the fluorescent reporter molecule forquantitative detection of its now unquenched signal using a real-timefluorescent detection system.

Variations on the TaqMan™ detection methodology that are also suitablefor use with the described invention include the use of dual-probetechnology (LightCycler™) or fluorescent amplification primers (Sunrise™technology). Both these techniques may be adapted in a manner suitablefor use with bisulfite treated DNA, and moreover for methylationanalysis within CpG dinucleotides.

Typical reagents (e.g., as might be found in a typical MethyLight™-basedkit) for MethyLight™ analysis may include, but are not limited to: PCRprimers for specific bisulfite sequences, i.e. bisulfite convertedgenetic regions (or bisulfite converted DNA or bisulfite converted CpGislands); probes (e.g. TaqMan® or LightCycler™) specific for saidamplified bisulfite converted sequences; optimized PCR buffers anddeoxynucleotides; and a polymerase, such as Taq polymerase. Thebisulfite conversion and amplification procedure according to theinvention may be used in combination with this detection method.

The fragments obtained by means of the amplification can carry adirectly or indirectly detectable label. Preferred are labels in theform of fluorescence labels, radionuclides, or detachable moleculefragments having a typical mass, which can be detected in a massspectrometer. Where said labels are mass labels, it is preferred thatthe labeled amplificates have a single positive or negative net charge,allowing for better detectability in the mass spectrometer. Thedetection may be carried out and visualized by means of, e.g., matrixassisted laser desorption/ionization mass spectrometry (MALDI) or usingelectron spray mass spectrometry (ESI).

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry(MALDI-TOF) is a very efficient development for the analysis ofbiomolecules (Karas & Hillenkamp, Anal Chem., 60:2299-301, 1988). Ananalyte is embedded in a light-absorbing matrix. The matrix isevaporated by a short laser pulse thus transporting the analyte moleculeinto the vapour phase in an unfragmented manner. The analyte is ionizedby collisions with matrix molecules. An applied voltage accelerates theions into a field-free flight tube. Due to their different masses, theions are accelerated at different rates. Smaller ions reach the detectorsooner than bigger ones. MALDI-TOF spectrometry is well suited to theanalysis of peptides and proteins. The analysis of nucleic acids issomewhat more difficult (Gut & Beck, Current Innovations and FutureTrends, 1:147-57, 1995). The sensitivity with respect to nucleic acidanalysis is approximately 100-times less than for peptides, anddecreases disproportionally with increasing fragment size. Moreover, fornucleic acids having a multiply negatively charged backbone, theionization process via the matrix is considerably less efficient. InMALDI-TOF spectrometry, the selection of the matrix plays an eminentlyimportant role. For desorption of peptides, several very efficientmatrixes have been found which produce a very fine crystallization.There are now several responsive matrixes for DNA, however, thedifference in sensitivity between peptides and nucleic acids has notbeen reduced. This difference in sensitivity can be reduced, however, bychemically modifying the DNA in such a manner that it becomes moresimilar to a peptide. For example, phosphorothioate nucleic acids, inwhich the usual phosphates of the backbone are substituted withthiophosphates, can be converted into a charge-neutral DNA using simplealkylation chemistry (Gut & Beck, Nucleic Acids Res. 23: 1367-73, 1995).The coupling of a charge tag to this modified DNA results in an increasein MALDI-TOF sensitivity to the same level as that found for peptides.

The amplificates may also be further detected and/or analysed by meansof oligonucleotides constituting all or part of an “array” or “DNA chip”(i.e., an arrangement of different oligonucleotides and/or PNA-oligomersbound to a solid phase). Such an array of different oligonucleotide-and/or PNA-oligomer sequences can be characterized, for example, in thatit is arranged on the solid phase in the form of a rectangular orhexagonal lattice. The solid-phase surface may be composed of silicon,glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, orgold. Nitrocellulose as well as plastics such as nylon, which can existin the form of pellets or also as resin matrices, may also be used. Anoverview of the Prior Art in oligomer array manufacturing can begathered from a special edition of Nature Genetics (Nature GeneticsSupplement, Volume 21, January 1999, and from the literature citedtherein). Fluorescently labeled probes are often used for the scanningof immobilized DNA arrays. The simple attachment of Cy3 and Cy5 dyes tothe 5′—OH of the specific probe are particularly suitable forfluorescence labels. The detection of the fluorescence of the hybridizedprobes may be carried out, for example, via a confocal microscope. Cy3and Cy5 dyes, besides many others, are commercially available. Thebisulfite conversion and amplification procedure according to theinvention may be used in combination with this detection method.

A particular preferred embodiment of the invention is a method forproviding a decontaminated nucleic acid for hybridisation on aDNA-Array, preferably an Oligonucleotide-Array, suitable for DNAmethylation analysis.

Of course, a particular preferred embodiment of the invention is also aimproved method for bilsulfite conversion of DNA. Thereby non-methylatedcytosines are converted to uracil while methylated cytosines remainunchanged. According to this embodiment, a nucleic acid is incubatedwith a bisulfite reagent containing solution, whereby the unmethylatedcytosines within said nucleic acid are sulfonated and/or deaminated butnot yet desulfonated as this is described above. Afterwards thesulfonated and/or deaminated template nucleic acid is mixed withcomponents required for a polymerase mediated amplification reaction oran amplification based detection assay. Thereafter the template nucleicacid is desulfonated by briefly incubating the mixture at an increasedtemperature. Subsequently the desulfonated template nucleic acid isamplified. In a particularly preferred variant the polymerase basedamplification reaction is started by a brief incubation at increasedtemperature (heat activation). Simultaneously, this brief incubation atincreased temperature serves to desulfonate the sulfonated and/ordeaminated template nucleic acid. Furthermore it is particularlypreferred that the polymerase is a heat stable polymerase.

This particular embodiment has the advantage, in comparison to knownmethods of bisulfite treatment, that the purification step afterbisulfite treatment becomes dispensable. This is a simplification whichresults in reduction of costs and handling effort, minimizes loss ofbisulfite treated DNA and is also time-saving. Therefore the use of thisembodiment is preferred if DNA samples are treated with bisulfite andsubsequently are amplified. This is especially preferred if large amountof samples are analyzed. The use of this embodiment is further preferredwith regard to sensitive detection methods for DNA methylation analysislike COBRA, MS-SNuPE, MSP, nested MSP, HeavyMethyl™ and MethyLight™.

Furthermore, the invention regards to a test kit for the realisation ofthe method according to the invention with a component containingbisulfite, for example a reagent or solution containing bisulfite, and acomponent containing an enzymatic activity. This enzymatic activityspecifically degrades DNA containing non-sulfonated uracil. Inparticular this enzymatic activity is an activity of a DNA-glycosylaseand/or an endonuclease, preferentially this enzymatic activity is anuracil-DNA-glycosylase, and more preferentially this enzymatic activityis uracil-N-DNA-Glycosylase (UNG). The added degrading enzyme activityis characterized by its ability to cause specifically apyrimidinic sitesand/or one or more nicks adjacent to a non-sulfonated uracil base. Inany case, this will result in a block of the replication by DNApolymerase. In a particular test kit, the enzymatic activity ischaracterized by cleaving the uracil base from the phosphodiesterbackbone of non-sulfonated uracil containing nucleic acid, but has noeffect on sulfonated uracil containing nucleic acid or on thyminecontaining nucleic acid, that does not contain uracil. The resultingapyrimidinic sites block replication by DNA polymerases, and are verylabile to acid/base hydrolysis.

A further test kit comprises one or more of the additional components.This can be:

-   -   one or more denaturing reagent and/or solution, for example:        dioxane or diethylene glycol dimethyl ether (DME) or any        substance, which is suitable as described in WO 05/038051;    -   one or more scavenger, for example        6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid or other        scavengers as described in WO 01/98528 or WO 05/038051;    -   one or more primers, which are suitable for the amplification of        one or more DNA amplificates, amongst others the primer or        primers can be modified, for example with a quencher and/or a        label for detection as well known by a person skilled in the art        like the dye FAM or the quencher BHQ black hole or dabcyl;    -   one or more probes, which can be any probe, which can be used to        specifically record the amplification of one or more        amplificates for example in a real-time-assay, amongst others        the probe or probes can be modified, for example with a        quenscher and/or a label for detection as well known by a person        skilled in the art like the dye FAM or the quencher BHQ black        hole or dabcyl;    -   one or more blockers, which are nucleic acids and can be used to        block the binding of a specific primer or the replication by DNA        polymerase, amongst others the blocker or blockers can be        modified, for example with a quenscher and/or a label for        detection as well known by a person skilled in the art like the        dye FAM or the quencher BHQ black hole or dabcyl;    -   one or more reaction buffers, which are suitable for a bisulfite        treatment and/or a PCR reaction,    -   nucleotides, which can be dATP, dCTP, dUTP and dGTP or any        derivative of these nucleotides,    -   MgCl₂ as a substance or in solution and/or any other magnesium        salt, which can be used to carry out a DNA polymerase        replication;    -   DNA polymerase, for example Taq polymerase or any other        polymerase with or without proof-reading acitivity,—dye or        quencher, which can be used for the detection of the        amplificates as known in the art, for example an intercalating        dye like SYBR Green or a dye for linkage to a primer or probe or        blocker like the dye FAM or the quencher BHQ black hole or        dabcyl; and/or    -   any reagent, solution, device and/or instruction which is useful        for realisation of an assay according to the invention.

The methods and test kits disclosed here are preferable used for thediagnosis and/or prognosis of adverse events for patients orindividuals, whereby diagnosis means diagnose of a adverse event, apredisposition for a adverse event and/or a progression of a adverseevents. hese adverse events belong to at least one of the followingcategories: undesired drug interactions; cancer diseases; CNSmalfunctions, damage or disease; symptoms of aggression or behavioraldisturbances; clinical, psychological and social consequences of braindamage; psychotic disturbances and personality disorders; dementiaand/or associated syndromes; cardiovascular disease, malfunction ordamage; malfunction, damage or disease of the gastrointestinal tract;malfunction, damage or disease of the respiratory system; lesion,inflammation, infection, immunity and/or convalescence; malfunction,damage or disease of the body as an abnormality in the developmentprocess; malfunction, damage or disease of the skin, of the muscles, ofthe connective tissue or of the bones; endocrine and metabolicmalfunction, damage or disease; headaches or sexual malfunction.

The methods and test kits also serve in a particularly preferred mannerfor distinguishing cell types, tissues or for investigating celldifferentiation. They serve in a particularly preferred manner foranalysing the response of a patient to a drug treatment.

In another preferred manner the methods and test kits of the inventioncan also be used to characterize the DNA methylation status in thatpositions are methylated or non-methylated compared to normal conditionsif a single defined disease exists. In a particular preferred mannerthey can serve for identifying an indication-specific target, wherein atemplate nucleic acid is treated with bisulfite and UNG enzyme activity,and wherein an indication-specific target is defined as differences inthe DNA methylation status of a DNA derived from a diseased tissue incomparison to a DNA derived from a healthy tissue. These tissue samplescan originate from diseased or healthy patients or from diseased orhealthy adjacent tissue of the same patient.

In a particular preferred manner the indication specific target is aprotein, peptide or enzyme, and in particular a per se known modulatorof the coded protein, peptide or enzyme is assigned with the specificindication of the diseased tissue. In a particular preferred manner thismodulator serves for preparing a pharmaceutical composition with aspecific indication, in particular a specific cancer indication.

In a particular preferred manner the enzyme UNG serves as an enzyme forgeneration of contamination free nucleic acids for methylation analysis.

Exemplary Preferred Embodiments:

Particular aspects provide an assay for providing a decontaminatednucleic acid suitable for DNA methylation analysis, comprising: a)contacting a DNA sample with a bisulfite reagent under conditionssuitable to produce a bisulfite-treated sulfonated DNA sample, whereinunmethylated cytosines of the DNA are sulfonated, sulfonated anddeaminated, or comprise a mixture of both forms, and wherein saidsulfonated, or sulfonated and deaminated uracil has not beensignificantly desulfonated; and b) further contacting thebisulfite-treated sulfonated DNA sample with an amount of an enzyme thatspecifically degrades any contaminating nonsulfonated-uracil-containingnucleic acids to provide for a decontaminated DNA sample. In particularaspects, the contaminating nonsulfonated-uracil-containing nucleic acidscomprise nonsulfonated-uracil-containing carry-over contaminants from aprior nucleic acid amplification reaction, and wherein thedecontaminated DNA sample is thereby optimized for use in a subsequentamplification analysis. Preferably, the subsequent amplificationanalysis comprises DNA methylation analysis.

Additional aspects provide the above methods, further comprising:—mixingthe bisulfite-treated sulfonated DNA sample with at least one componentrequired for a polymerase-mediated amplification reaction or anamplification-based detection assay;—inactivating, after specificallydegrading any nonsulfonated-uracil-containing nucleic acids, thespecifically degrading enzymatic activity; and—desulfonating, after saidinactivating, the bisulfite-treated sulfonated DNA sample. In particularembodiments, inactivating and desulfonating occur simultaneously byincubating the mixture at an increased temperature, said incubationsufficient to inactivate the specifically degrading enzymatic activity,and desulfonate the bisulfite-treated sulfonated DNA. Preferably, themethod further comprises, after desulfonating, amplifying thedesulfonated DNA. In particular embodiments, the specifically degradingenzyme is at least one selected from the group consisting of a DNAglycosylase and an endonuclease. Preferably, the DNA glycosylase isuracil-DNA-glycosylase (UNG).

Additional embodiments further comprise, after inactivating anddesulfonating, performing a polymerase-based amplification reaction oran amplification-based assay. In particular aspects, thepolymerase-based amplification reaction is initiated by a briefincubation at increased temperature, said incubation sufficient toinactivate the specifically degrading enzymatic activity. Preferably,the polymerase is a heat stable polymerase. In preferred aspects, thepolymerase-mediated amplification, or the amplification-based assay isperformed in the presence of dUTPs instead of dTTPs.

In preferred embodiments, the amount of the specifically degradingenzyme is sufficient to degrade substantially all of anynonsulfonated-uracil-containing nucleic acid contaminants.

Yet further embodiments provide a method for bisulfite treatment of anucleic acid, comprising: a) contacting a nucleic acid with a bisulfitereagent under conditions suitable to produce a bisulfite-treatedsulfonated nucleic acid sample, wherein unmethylated cytosines of thenucleic acid are sulfonated, sulfonated and deaminated, or comprise amixture of both forms, and wherein said sulfonated, or sulfonated anddeaminated uracil has not been significantly desulfonated; b) furthercontacting the bisulfite-treated sulfonated nucleic acid sample with atleast one component required for a polymerase-mediated amplificationreaction or an amplification-based detection assay; and c) desulfonatingthe bisulfite-treated sulfonated nucleic acid sample by a briefincubation of the mixture at an increased temperature. Particularembodiment of this method further comprise, after desulfonating,amplifying the desulfonated nucleic acid with a polymerase-mediatedamplification reaction or an amplification-based detection assay.Preferably, a heat stable polymerase is used. In particular embodiments,the polymerase-based amplification reaction is briefly incubated at anincreased temperature to simultaneously start the reaction, and allowfor desulfonation of the bisulfite-treated sulfonated nucleic acidsample.

Further aspects provide a kit for decontaminating a nucleic acid sample,comprising: a bisulfite reagent; and an enzymatic activity thatspecifically degrades nonsulfonated-uracil-containing DNA. Preferably,the enzymatic activity is that of uracil-DNA-glycosylase (UNG). Inadditional embodiments, the kit further comprises at least one componentselected from the group consisting of: denaturing reagent and solution;scavenger; primer; probe; reaction buffer; nucleotides; MgCl₂ solution;polymerase, dye for the production of amplificates; and instructions forusing the kit.

Additional embodiments provide a method for diagnosis, prognosis, orboth of an adverse event for patients or individuals, comprising use ofthe above described methods or kits, and wherein the adverse event is atleast one selected from the group consisting of: undesired druginteractions; cancer diseases; CNS malfunctions; damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damages; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease of the gastrointestinal tract;malfunction, damage or disease of the respiratory system; lesion,inflammation, infection, immunity and/or convalescence; malfunction,damage or disease of the body as an abnormality in the developmentprocess; malfunction, damage or disease of the skin, of the muscles, ofthe connective tissue or of the bones; endocrine and metabolicmalfunction, damage or disease; headaches and sexual malfunction.

Yet further embodiments provide a method for distinguishing cell typesor tissue or for investigating cell differentiation, comprisingperforming a nucleic acid-based assay suitable for distinguishing celltypes or tissue or for investigating cell differentiation, and whereinone or more of the above described methods or kits are used to providefor decontaminated nucleic acid samples.

Additional aspects provide a method for identifying anindication-specific target or marker, comprising performing a suitableDNA methylation assay on DNA of a test sample and determining themethylation status, relative to a normal control status, at one or morepositions of the DNA, wherein the determined methylation status isindicative for the presence or absence of a single defined disease, andwherein one or more of the above described methods are used todecontaminate the test sample DNA prior to performing the methylationanalysis. In particular embodiments, the methods comprise: treating atest nucleic acid sample with a bisulfite reagent to sulfonateunmethylated cytosines of the nucleic acid; contacting thebisulfite-treated nucleic acid with uracil-DNA-glycosylase (UNG) underconditions suitable to specifically degrade any contaminatingnonsulfonated-uracil-containing nucleic acids to provide for adecontaminated nucleic acid sample; and performing a methylation assayon the decontaminated nucleic acid to determine the methylation status,relative to a normal control status, at one or more positions of theDNA, wherein the determined methylation status is indicative for thepresence or absence of a single defined disease. In particular aspects,the indication-specific target is a protein, peptide or enzyme.Preferably, a per se known modulator of the coded protein, peptide orenzyme is assigned to the specific indication of the diseased tissue.

Additional aspects provide a method for providing decontaminated nucleicacid suitable for methylation analysis, comprising bisulfite treatmentof a nucleic acid sample to provide for a sulfonated nucleic acidsample; and contacting the bisulfite-treated sulfonated nucleic acidsample with uracil-DNA-glycosylase (UNG) under conditions suitable tospecifically degrade any contaminating nonsulfonated-uracil-containingnucleic acids to provide for a decontaminated nucleic acid samplesuitable for methylation analysis.

EXAMPLE 1 (Amplification of Methylated DNA of the GSTP1 Gene (Also Knownas GST-pi Gene) Wherein Human DNA Containing Sulfonated Uracils Servedas Template)

The use of uracil-DNA-glycosylase is a method well known in the art toavoid false positive results in polymerase based amplification methods,caused by cross contamination by previously amplified products (Pang J.,Mol Cell Probes. 1992 Jun;6(3):251-6). This method is however notapplicable for polymerase based amplification methods which have thepurpose to detect uracil bases within the given template. This is thecase in DNA methylation analysis, wherein one way to detect thedifference between methylated and unmethylated cytosines is to mirrorthese differences into the difference between cytosine and uracil, whichis facilitated by the widely spread use of common bisulfite conversionmethods. These have the effect to convert unmethylated cytosines intouracils whereas methylated cytosines remain cytosines. Therefore insubsequent amplification reactions to detect methylation patterns thetemplate contains uracils.

In the following example it was shown that the method according to theinvention allows Uracil-DNA-glycosylase (UNG) based technique for carryover prevention of bisulfite converted DNA, without loss of the criticalinformation, which bases were unmethylated and which were methylated. Toachieve this the following steps were carried out:

Two nucleic acid samples, containing 1.5 μg GpGenome™ UniversalMethylated DNA (Chemicon International) diluted in 100 μl water weremixed with 354 μl of bisulfite solution (5.89 mol/l) and 146 μl ofdioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated with the following temperature program for atotal of 5 h: 30 min at 50° C.; a first thermospike (99.9° C.) for 3min; 1.5 h at 50° C.; a second thermospike (99.9° C.) for 3 min; 3 h at50° C. One of the reaction mixtures served as a control whereas theother was treated according to the invention. The reaction mixtures ofboth the control and the test reaction were subsequently purified byultrafiltration by means of a Millipore Microcon column. Thepurification was conducted essentially according to the manufacturer'sinstructions. For this purpose, the reaction mixture was mixed with 200μl of water, loaded onto the ultrafiltration membrane, centrifuged for15 min and subsequently washed with water. The DNA remains on themembrane in this treatment. For the control sample an alkalinedesulfonation was performed according to the methods, which are state ofthe art (see for example US 20040152080, 20040115663, WO 2004/067545).For this purpose, 100 μl of a 0.2 mol/l NaOH was added and incubated for10 min. For the other sample this desulfonation step was replaced byadding 100 μl water. A centrifugation (10 min) was then conducted,followed by a final washing step with water. After this, the DNA waseluted. For this purpose, the membrane was mixed for 10 minutes with 50μl of warm 1×TE buffer (50° C.) adjusted to pH7. The membrane was turnedover according to the manufacturer's instructions. Subsequently arepeated centrifugation was conducted, with which the DNA was removedfrom the membrane.

Subsequently the DNA was stored at 4° C. for 12 h and then used astemplate in a PCR reaction.

By stopping the chemical reaction after sulfonation all unmethylatedcytosines are converted into C6 sulfonated uracils(5,6-Dihydro-6-sulfonyl-uracil) and methylated cytosines remainunchanged. However after a complete desulfonation, as described in theart, all unmethylated cytosines are converted in uracil and methylatedcytosines remain unchanged.

As a control for the UNG activity 10⁵ copies of a uracil containing PCRproduct were added to the reaction premix, generated by use of the sameprimers but under presence of dUTP instead of dTTP. Reamplification tookplace when UNG was absent, with the expected efficiency of a crossingpoint of 22,6. However when UNG was present in the PCR-mix the crossingpoints reached only a value of 35,8 (FIG. 4). This differencedemonstrates nicely the efficient degradation of PCR products by UNGresulting from the cleavage of uracils out of the DNA. In this exampleit was shown that desulfonated conventionally bisulfite treated DNA isalso degraded by UNG (FIG. 2). However, not desulfonated bisulfitetreated DNA does not work as substrate for UNG and serves—even afterpreincubation with UNG—as working template in an amplification reaction(FIG. 3).

In the Example the desulfonation of template DNA required for thesuccessful amplification took place during the initial denaturing phaseof the PCR reaction at 95° C. Only precondition for this step is analkaline pH, such as given in the utilized PCR buffer. Simultaneouslythe UNG activity is terminated and is hence not capable of cleaving ordegrading newly generated PCR product anymore.

In this Example three different concentrations (10 ng, 0.1 ng and 0.1ng) of each desulfonated and sulfonated (containing 6-sulfonated5,6-dihydro-uracils) DNA were used as templates in two differentHot-Start PCR reactions.

In one case the reaction mix contained 0.2 Units UNG, in the other caseno UNG was added. PCR reactions were performed in the LightCycler™ in 20μl reaction volume and contained:

10 μl of template DNA (in different concentrations)

2 μl of FastStart LightCycler™ Mix for Hybridization probes (RocheDiagnostics)

3.5 mM MgCl₂ (Roche Diagnostics)

0.30 μM forward primer (SEQ ID NO:1, TIB-MolBiol)

0.30 μM reverse primer (SEQ ID NO:2, TIB-MolBiol)

0.15 μM Probe1 (SEQ ID NO:3, TIB-MolBiol)

0.15 μM Probe2 (SEQ ID NO:4, TIB-MolBiol)

optional 0.2 Unit Uracil-DNA-Glycosylase (Roche Diagnostics)

The temperature-time-profile was programmed as follows:

Pre-incubation (UNG active) 15 min by 25° C.

Activation of polymerase: 20 min by 95° C.

50 temperature cycles: 10 sec by 95° C.

30 sec at 56° C.

10 sec at 72° C.

Finally the reaction is cooled down to 35° C.

The primers (SEQ ID NO:1, SEQ ID NO:2) used amplify a 123 bp longfragment of the GSTP1 gene (SEQ ID NO:5, nt 1184 to nt 1304 in GenbankAccession X08058). By utilizing sequence specific hybridization probes(SeqID 3, SEQ ID NO:4) the amplification rate was detected in a RealTime PCR. Data interpretation was carried out via the LightCyclerSoftware in channel F2/F1.

The crossing point (Cp) was generated automatically by employing themethod “Second Derivative Maximum” (Table 1).

RESULTS. The results of the experiment are summarized in Table 1. Thereamplification of 10⁵ copies of uracil containing amplicons results inCT of 22,6 without UNG and 35,8 with UNG. The CT delay of 13 cyclesdemonstrates the efficient degradation of uracil containing template bythe glycosylase. Also desulfonated bisulfite converted DNA was degradedby UNG and no amplification was measurable in the reaction with UNG. Inthe reaction without UNG the 10,1, and 0,1 ng DNA was detected at CT of28,5/31,7/33,8. In contrast to this, sulfonated DNA, prepared accordingto the invention, was amplified in both cases, without and withUracil-DNA-Glycosylase with almost the same efficiency and were detectedat CT of 29,3/32,2/34,1 and 29,9/32,7/34,8 respectively.

TABLE 1 Crossing point Crossing point of Template of reaction reactionwith 0.2 DNA DNA in ng without UNG Unit UNG added PCR Amplicons 10⁵copies 22.6 35.8 containing Uracil desulfonated DNA 10 28.5 no signal 131.7 no signal 0.1 33.8 no signal sulfonated DNA 10 29.3 29.9 1 32.232.7 0.1 34.1 34.8

TABLE 2 Sequences of Oligonucleotides SeqID Name Sequence SEQ IDGSTP1.10F1 GGGAttAtttTTATAAGGtT NO: 1 SEQ ID GSTP1.10R5TaCTaaaAaCTCTaAaCCCCATC NO: 2 SEQ ID GSTP1.10-TTCGtCGtCGtAGTtTTCGtt-Fluo NO: 3 fluo1 SEQ ID GSTP1.10-red640-tAGTGAGTACGCGCGGtt- NO: 4 red 1 PH Seq ID GSTP15′GGGAttAtttTTATAAGGtTCGGAG NO: 5 amplicon GtCGCGAGGttTTCGtTGGAGTTTCGtCGtCGtAGTtTTCGttAttAGTGAGTA CGCGCGGttCGCGTtttCGGGGATGGGGtTtAGAGtTtttAGtAFluo=fluoresceine label, red640=LightCycler™ fluorescence label forchannel F2, PH=3′OH-Phosphorylation. Small written t's point toconverted cytosines by bisulfite treatment, respectively small a's pointto the complementary adenosine bases in the reverse complementsynthesized strand.

EXAMPLE 2 (The Stability of the Sulfonated Nucleic Bases in the Presenceof UNG Activity was Analyzed When Stored at 4° C. or 40° C.)

In this experiment the stability of the sulfonated nucleic bases in thepresence of UNG activity was analyzed when stored at 4° C. or 40° C.Again, two nucleic acid samples, containing 1.5 μg GpGenome™ UniversalMethylated DNA (Chemicon International) diluted in 100 μl water weremixed with 354 μl of bisulfite solution (5.89 mol/l) and 146 μl ofdioxane containing a radical scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated with the following temperature program for atotal of 5 h: 30 min at 50° C.; a first thermospike (99.9° C.) for 3min; 1.5 h at 50° C.; a second thermospike (99.9° C) for 3 min; 3 h at50° C. One of the reaction mixtures served as a control whereas theother was treated according to the invention. The reaction mixtures ofboth the control and the test reaction were subsequently purified byultrafiltration by means of a Millipore Microcon column. Thepurification was conducted essentially according to the manufacturer'sinstructions. For this purpose, the reaction mixture was mixed with 200μl of water, loaded onto the ultrafiltration membrane, centrifuged for15 min and subsequently washed with water. The DNA remains on themembrane in this treatment. For the control sample an alkalinedesulfonation was performed according to the methods, which are state ofthe art (see for example US 20040152080, 20040115663, WO 2004/067545)(named ‘desulfonated’ in table 3). For this purpose, 100 μl of a 0.2mol/l NaOH was added and incubated for 10 min. For the other sample thisdesulfonation step was replaced by adding 100 μl water (named‘sulfonated’ in table 3). A centrifugation (10 min) was then conducted,followed by a final washing step with water. After this, the DNA waseluted. For this purpose, the membrane was mixed for 10 minutes with 50μl of warm 1×TE buffer (50° C.) adjusted to pH7. The membrane was turnedover according to the manufacturer's instructions. Subsequently arepeated centrifugation was conducted, with which the DNA was removedfrom the membrane.

Subsequently the DNA was divided in aliquots and some of them werestored at 4° C. for 12 h, others at 4° C. for 144 h and then used astemplate in a PCR reaction. To show the robustness of the methodaccording to the invention applicants wanted to analyze whether thisprotecting effect of sulfonation would be stable over a period of time.The PCR reaction was performed under the same conditions.

In addition aliquots were stored at an increased temperature of 40° C.for 22 h and then used as template in a PCR reaction.

By stopping the chemical reaction after sulfonation the unmethylatedcytosines were converted into C6 sulfonated uracils(5,6-Dihydro-6-sulfonyl-uracil) and methylated cytosines remainedunchanged. However after a complete desulfonation, as described in theart, all unmethylated cytosines would have converted into uracil insteadand methylated cytosines remain unchanged.

As a control for the UNG activity 10⁵ copies of a uracil containing PCRproduct were added to the reaction premix, generated by use of the sameprimers but under presence of dUTP instead of dTTP. Reamplification tookplace when UNG was absent, with the expected efficiency of a crossingpoint of 22,6. However, when UNG was present in the PCR-mix the crossingpoints reached only a value of 35,1 (Table 3). This differencedemonstrates nicely the efficient degradation of PCR products by UNGresulting from the cleavage of uracils out of the DNA.

In this Example it was shown that bisulfite treated DNA which is notdesulfonated according to the invention is stable at 4° C. for a longerperiod of at least 144 hrs. In addition it was shown that even storageat 40° C. for a period of 22 hrs does not have a major effect on the UNGprotecting effect of sulfonation at C6-uracils.

TABLE 3 Crossing point of Crossing point of reaction Template reactionwith 0.2 Unit DNA in ng without UNG UNG added DNA after 12 h at 4° C.PCR amplicons 10⁵ copies 22.6 35.8 containing uracil desulfonated 0.133.8 no signal DNA sulfonated DNA 0.1 34.1 34.8 DNA after 144 h at 4° C.PCR amplicons 10.00E5 22.6 35.1 containing uracil copies desulfonated 1028.3 no signal DNA desulfonated 1 30.8 no signal DNA desulfonated 0.133.5 no signal DNA sulfonated DNA 10 28.7 29.5 sulfonated DNA 1 31.731.8 sulfonated DNA 0.1 33.7 34.2 DNA after 22 h at 40° C. PCR amplicons10.00E5 22.6 35.7 containing uracil copies desulfonated 10 29.5 nosignal DNA desulfonated 1 32.5 no signal DNA desulfonated 0.1 34.9 nosignal DNA sulfonated DNA 10 29.5 30.5 sulfonated DNA 1 32.5 33.3sulfonated DNA 0.1 34.6 36.1

EXAMPLE 3 (Comparison of Particular Inventive Methods with the StandardWorkflow by Means of the Determination of the Methylation Rate of theTPEF Gene (Also Known as TMEFF2) in Colon Cancer Tissue)

1 μg of genomic DNA (200 μl) was extracted from tumours and normaladjacent tissue of 12 patients with colon cancer, respectively. The 24samples obtained in this way were each divided into 2×100 μl DNA. 100 μlof each sample was treated according to standard procedures (bisulfitetreatment protocol A, sample set A) or to the method according to theinvention (bisulfite protocol B, sample set B). In between the DNA wasstored at −20° C.

Standard Workflow:

Sample Set A:

Measurement of the DNA was performed according to the C3 quantificationassay version A and according to the HeavyMethyl™ assay for the TPEFgene version A. A standard A was generated for calibration.

Generation of Standard A:

5 tubes each with 2 μg universal methylated DNA were treated withbisulfite according to the bisulfite treatment protocol A and pooledafterwards. The concentration of the DNA in solution was determined bymeans of UV at 260 nm after the bisulfite reaction.

Bisulfite Treatment Protocol A (Standard Procedures):

100 μl of the samples (sample set A) containing 0.5 μg DNA diluted in100 μl water were mixed with 354 μl of bisulfite solution (5.89 mol/l)and 146 μl of dioxane containing a scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated with the following temperature program for atotal of 5 h: 30 min 50° C.; one thermospike 99.9° C. for 3 min; 1.5 h50° C; one thermospike 99.9° C. for 3 min; 3 h 50° C. The DNA of thereaction mixtures was subsequently purified by ultrafiltration by meansof a Millipore Microcon column. The purification was conductedessentially according to the manufacturer's instructions. For thispurpose, the reaction mixture was mixed with 200 μl of water, loadedonto the ultrafiltration membrane, centrifuged for 15 min andsubsequently washed with water. The DNA remains on the membrane in thistreatment. For complete desulfonation 100 μl of a 0.2 mol/l NaOHsolution was added and incubated for 10 min. A centrifugation for 10 minwas then conducted, followed by a final washing step with water. Afterthis, the DNA was eluted. For this purpose, the membrane was mixed for10 minutes with 75 μl of prewarmed 1×TE buffer (50° C.) adjusted to pH8.5. Then the membrane was turned over and centrifuged according to themanufacturer's instructions to recover the DNA from the membrane.

C3 quantification assay:

The C3 quantification assay is a quantification assay specific for thetotal amount of bisulfite converted DNA. The assay amplifies a fragmentof DNA that comprises multiple cytosine (but not CpG) positions in thegenomic form, which are initially converted to uracil and duringamplification replaced by thymine in the bisulfite converted variant.Accordingly the assay does not quantify for unconverted or partiallyconverted bisulfite treated DNA (i.e. wherein the target sequencecomprises one or more cytosine positions which have not been convertedto thymine). The quantity of DNA in the sample is deduced by comparisonof the measured CP (crossing point, which represents the thresholdcycle) to a standard curve relating such CP values to DNA amounts. Thestandard curve is based on measurements of known quantities of bisulfiteconverted DNA with the according assay.

C3 Quantification Assay Version A:

A 20 μl reaction mixture contained:

-   -   2 μl of template DNA    -   2 μl of FastStart LightCycler™ Mix for hybridisation probes        (Roche Diagnostics)    -   3.5 mmol/l MgCl₂ (Roche Diagnostics)    -   0.60 μmol/l forward primer SEQ ID NO:6, TIB-MolBiol)    -   0.60 μmol/l reverse primer (SEQ ID NO:7, TIB-MolBiol)    -   0.2 μmol/l probe 1 (SEQ ID NO:8, TIB-MolBiol)        The assay was performed according to the following        temperature-time-profile:    -   activation 10 min at 95° C.    -   50 cycles: 10 sec at 95° C.        -   30 sec at 56° C.        -   10 sec at 72° C.

The used primers (SEQ ID NO:6 and SEQ ID NO:7) amplify a fragment of 123bp of the GSTP1 gene (SEQ ID NO:9. nucleotide 2273 to nucleotide 2402 ofGenBank Accession Number X08058). The detection was carried out duringthe annealing phase at 56° C. in channel F1 at 530 nm. The crossingpoints (CP) were calculated according to the “second derivative maximum”method by means of the LightCycler™ software.

Detection of the Methylation Rate According to the HeavyMethyl™ Assayfor the TPEF Gene Version A:

A 20 μl reaction mixture contained:

-   -   2 μl of template DNA    -   2 μl of FastStart LightCycler™ Mix for hybridization probes        (Roche Diagnostics)    -   3.5 mmol/l MgCl₂ (Roche Diagnostics)    -   0.30 μmol/l forward primer (SEQ ID NO:10, TIB-MolBiol)    -   0.30 μmol/l reverse primer (SEQ ID NO:11, TIB-MolBiol)    -   4.0 μmol/l blocker (SEQ ID NO:12, TIB-MolBiol)    -   0.15 μmol/l hybridization probe (SEQ ID NO:13, TIB-MolBiol)    -   0.15 μmol/l hybridization probe (SEQ ID NO:14, TIB-MolBiol)        The assay was performed according to the following        temperature-time-profile:    -   activation 10 min at 95° C.    -   50 cycles: 10 sec at 95° C.        -   30 sec at 56° C.        -   10 sec at 72° C.

The used primers (SEQ ID NO:10 and SEQ ID NO:11) amplify a fragment of113 bp of the TPEF gene (SEQ ID NO:15. nucleotide 1102 to nucleotide1214 of GenBank Accession Number AF242221). The detection was carriedout during the annealing phase at 56° C. in channel F2/F1 at 640/530 nm.The crossing points (CP) were calculated according to the “secondderivative maximum” method by means of the LightCycler™ software.

Calculation of DNA Amounts from CP:

Both the C3 quantification assay and HeavyMethyl assay for the TPEF geneare Real Time PCR assays using an external standard for calculating theDNA amount of the measured samples. The absolute value (ng) for anunknown concentration is obtained by a comparison of the amplificationof DNA in an unknown sample against a standard curve prepared with knownconcentrations of the same target. The standard samples are amplified inseparate capillaries but within the same LightCycler™ run. The standardcurve is the linear regression line through the data points on a plot ofcrossing points (threshold cycle) versus logarithm of standard sampleconcentration. The absolute amount of DNA (ng) of the unknown samplematches the data point of the standard curve at which the CP of theunknown sample fits the standard curve.

TABLE 4 Sequence of Oligonucleotides SeqID Name Sequence SEQ ID NO: 6C3F GGAGTGGAGGAAAtTGAGAt SEQ ID NO: 7 C3R CCACACAaCAaaTaCTCAaAaC SEQ IDNO: 8 C3-TAQ FAMTGGGTGTTTGTAATTTTTGTTTTG TGTTAGGTT-BHQ1 SEQ ID NO: 9 C3-GGAGTGGAGGAAAtTGAGAtttAtTGA amplicon GGTTACGTAGTTTGtttAAGGTtAAGttTGGGTGttTGtAATttTTGtttTGTG ttAGGtTGttTtttAGGTGTtAGGTGAGtTtTGAGtAttTGtTGTGTGG SEQ ID NO: 10 TPEF-61S aAAAaAaAAAaaCTCCTCTaCATACSEQ ID NO: 11 TPEF-62S GGTtAtTGttTGGGttAAtAAATG SEQ ID NO: 12 TPEF-6B2aCATACaCCaCaaaTaaaTTaCCaaaA aCATCaaCCaa-PH SEQ ID NO: 13 TPEF-6SF1tTttttTTttCGGACGtCGtT-Fluo SEQ ID NO: 14 TPEF-6SR1red640-tCGGtCGATGtTttCGGtA A-PH SEQ ID NO: 15 TPEF-GGTtAtTGttTGGGttAAtAAATGGAG amplicon ttCGtTtTttttTTttCGGACGTCGtTGttCGGtCGATGtTttCGGtAAtttAt tCGCGGCGTATGtAGAGGAGttTTTtT tTTTtFluo=fluoresceine label, red640=LightCycler fluorescence label forchannel F2, PH=3′OH-Phosphorylation, FAM=5′-FAM label,BHQ1=BlackHoleQuencher1. Small written t's point to converted cytosinesby bisulfite treatment, respectively small a's point to thecomplementary adenosine bases in the reverse complement synthesizedstrand.Exemplary Method According to the Invention:Sample Set B:

Measurement of the DNA was performed according to the C3 quantificationassay version B and according to the HeavyMethyl assay for the TPEF geneversion B in addition of 10,000 copies of a PCR product of methylatedDNA. A standard B (C6 sulfonated uracil containing DNA) was generatedfor calibration.

Generation of Standard B (C6 Sulfonated Uracil Containing DNA):

5 tubes each with 2.0 μg universal methylated DNA were treated withbisulfite according to the bisulfite treatment protocol B. Theconcentration of the DNA in solution was determined by means of UV at260 nm after the bisulfite reaction.

Generation of PCR Products.

10 ng methylated bisulfite converted DNA generated according to standardprocedures (bisulfite protocol A) were amplified by means of theHeavyMethyl assay for the TPEF gene version A. The PCR products werepurified with the QIAquick PCR Purification Kit and subsequentlyanalyzed on a 2% agarose gel. After this, a serial dilution was carriedout with water to a final dilution of 1:10¹⁰. 2 μl of this dilution wasreamplified and quantificated according to the HeavyMethyl™ assay forthe TPEF gene version A. The copy number was determined: 2 μl of thesaid dilution contain 10,000 copies of PCR product.

Bisulfite Treatment Protocol B (Protocol for Carry Over Protection):

100 μl of the samples (sample set B) containing 0.5 μg DNA diluted in100 μl water were mixed with 354 μl of bisulfite solution (5.89 mol/l)and 146 μl of dioxane containing a scavenger(6-hydroxy-2,5,7,8-tetramethylchromane 2-carboxylic acid, 98.6 mg in 2.5ml of dioxane). The reaction mixture was denatured for 3 min at 99° C.and subsequently incubated with the following temperature program for atotal of 5 h: 30 min 50° C.; one thermospike 99.9° C. for 3 min; 1.5 h50° C.; one thermospike 99.9° C. for 3 min; 3 h 50° C. The DNA of thereaction mixtures was subsequently purified by ultrafiltration by meansof a Millipore Microcon column. The purification was conductedessentially according to the manufacturer's instructions. For thispurpose, the reaction mixture was mixed with 200 μl of water, loadedonto the ultrafiltration membrane, centrifuged for 15 min andsubsequently washed with water. The DNA remains on the membrane in thistreatment. In contrast to the bisulfite treatment protocol A the DNA wasnot incubated with NaOH, but additionally washed with water. After this,the DNA was eluted. For this purpose, the membrane was mixed for 10minutes with 75 μl of prewarmed water (50° C.). Then the membrane wasturned over and centrifuged according to the manufacturer's instructionsto recover the DNA from the membrane.

C3 Quantification Assay Version B:

A 20 μl reaction mixture contained:

-   -   2 μl of template DNA    -   2 μl PCR product (10,000 copies)    -   2 μl of FastStart LightCycler™ Mix for hybridization probes        (Roche Diagnostics)    -   3.5 mmol/l MgCl₂ (Roche Diagnostics)    -   0.60 μmol/l forward primer (SEQ ID NO:6, TIB-MolBiol)    -   0.60 μmol/l reverse primer (SEQ ID NO:7, TIB-MolBiol)    -   0.2 μmol/l probe 1 (SEQ ID NO:8, TIB-MolBiol)    -   0.2 units uracil-DNA-glycosylase (Roche Diagnostics)        The assay was performed according to the following        temperature-time-profile:    -   preincubation 10 min at 37° C.    -   desulfonation/activation 30 min at 95° C.    -   50 cycles: 10 sec at 95° C.        -   30 sec at 56° C.        -   10 sec at 72° C.            The used primers (SEQ ID NO:6 and SEQ ID NO:7) amplify a            fragment of 123 bp of the GSTP1 gene (SEQ ID NO:9.            nucleotide 2273 to nucleotide 2402 of GenBank Accession            Number X08058). The detection was carried out during the            annealing phase at 56° C. in channel F1 at 530 nm. The            crossing points (CP) were calculated according to the            “second derivative maximum” method by means of the            LightCycler™ software.            Detection of the Methylation Rate According to the            HeavyMethyl™ Assay for the TPEF Gene Version B:

A 20 μl reaction mixture contained:

-   -   2 μl of template DNA    -   2 μl PCR product (10,000 copies)    -   2 μl of FastStart LightCycler™ Mix for hybridisation probes        (Roche Diagnostics)    -   3.5 mmol/l MgCl₂ (Roche Diagnostics)    -   0.30 μmol/l forward primer (SEQ ID NO:10, TIB-MolBiol)    -   0.30 μmol/l reverse primer (SEQ ID NO:11, TIB-MolBiol)    -   4.0 μmol/l blocker (SEQ ID NO:12, TIB-MolBiol)    -   0.15 μmol/l hybridisation probe (SEQ ID NO:13, TIB-MolBiol)    -   0.15 μmol/l hybridisation probe (SEQ ID NO:14, TIB-MolBiol)    -   0.2 units uracil-DNA-glycosylase (Roche Diagnostics)        The assay was performed according to the following        temperature-time-profil:    -   preincubation 10 min at 37° C.    -   desulfonation/activation 30 min at 95° C.    -   50 cycles: 10 sec at 95° C.        -   30 sec at 56° C.        -   10 sec at 72° C.

The used primers (SEQ ID NO:10 and SEQ ID NO:11) amplify a fragment of113 bp of the TPEF gene (SEQ ID NO:15, nucleotide 1102 to nucleotide1214 of GenBank Accession Number AF242221). The detection was carriedout during the annealing phase at 56° C. in channel F2/F1 at 640/530 nm.The crossing points (CP) were calculated according to the “secondderivative maximum” method by means of the LightCycler™ software.

Calculation of DNA Amounts from CP:

Both the C3 quantification assay and HeavyMethyl™ assay for the TPEFgene are Real Time PCR assays using an external standard for calculatingthe DNA amount of the measured samples. The absolute value (ng) for anunknown concentration is obtained by a comparison of the amplificationof DNA in an unknown sample against a standard curve prepared with knownconcentrations of the same target. The standard samples are amplified inseparate capillaries but within the same LightCycler™ run. The standardcurve is the linear regression line through the data points on a plot ofcrossing points (threshold cycle) versus logarithm of standard sampleconcentration. The absolute amount of DNA (ng) of the unknown samplematches the data point of the standard curve at which the CP of theunknown sample fits the standard curve.

Calculation of the methylation rate from DNA amounts: The results of thestudy are presented as methylation rates of the promotor region of theTPEF gene. According to the PMR value method ( Eads CA et al. CancerRes., 61:3410-8, 2001. PMID: 11309301) the methylation rate is equal tothe percentage of methylated copies measured in a sample as proportionof the total DNA measured in the same sample. In table 5 and 6 all CPsreceived from the C3 and the TPEF assay and the resulting DNA amountsare listed. In the right column the methylation rate (PMR) is shown,which was calculated from the DNA amounts listed in colums before.

Results:

TABLE 5 Results from the standard workflow. Colon cancer and normaladjacent tissue samples were bisulfite treated with bisulfite treatmentprotocol A followed by quantification with the C3 quantification assayversion A using a calibration curve made by means of standard A. Thetable shows the crossing points and the calculated DNA amount of 2replicates. The HeavyMethyl ™ assay for the TPEF gene version A detectsonly methylated DNA from the promoter region of TPEF gene. The tableshows the measured CP values of 2 replicates and the calculated DNAamount. Finally the methylation percentages (PMR) were calculated by theratio of methylated DNA and total DNA. HeavyMethyl Assay C3Quantification Assay TPEF gene Version A Version B CP CP ng/PCR CP CPng/PCR Sample Type 1^(st) run 2^(nd) run mean 1^(st) run 2^(nd) run meanPMR % standard A   20 ng 25.86 25.64 26.28 26.55 standard A   5 ng 28.2927.72 28.51 28.24 standard A   5 ng 28.44 27.74 28.51 28.15 standard A1.25 ng 30.27 30.2 29.91 30.46 standard A 1.25 ng 30.23 30.41 29.9230.21 standard A 0.31 ng 32.48 32.01 31.46 31.51 standard A 0.31 ng31.89 32.18 31.13 31.54 1 normal 31.49 32.6 0.4 27.27 28.68 7.4 5% 2tumor — — 0.0 28.22 29.49 4.1 0% 3 normal 32.74 33.56 0.1 27.84 28.975.4 2% 4 tumor 29.51 30.96 1.5 28.08 29.42 4.4 33%  5 normal 31.97 33.030.3 27.72 29.09 5.6 5% 6 tumor 27.8 29.65 4.0 27.45 29.16 6.2 65%  7normal 32.02 33.47 0.2 27.62 28.93 6.0 4% 8 tumor 28.5 30.53 2.5 27.5629.2 5.9 43%  9 normal 32.47 33.8 0.1 28.06 29.13 4.7 3% 10 tumor 29.8731.17 1.2 27.91 29.07 5.1 23%  11 normal 31.93 33.65 0.2 27.17 29.12 7.23% 12 tumor 32.07 33.27 0.2 27.1 28.29 8.6 3% 13 normal 31.45 33.41 0.327.52 28.61 6.7 5% 14 tumor 29.87 30.64 1.3 27.83 28.44 6.2 22%  15normal 35.97 32.94 0.1 27.8 28.2 6.7 1% 16 tumor 28.92 29.07 2.8 28.5428.73 4.4 64%  17 normal 32.49 34.58 0.1 27.98 29.45 4.6 3% 18 tumor27.05 27.93 7.5 27.16 28.08 8.8 84%  19 normal 31.6 32.78 0.3 27.6 28.956.0 6% 20 tumor 28.78 30.11 2.4 27.59 28.79 6.2 38%  21 normal 35.2335.88 0.0 28.07 29.32 4.5 0% 22 tumor 35 36.93 0.0 27.91 29.08 5.1 0% 23normal 31.86 32.72 0.3 27.63 28.31 6.9 4% 24 tumor 30.01 31.55 1.0 27.4728.58 6.9 15%  neg. contr. — — — — — — —

TABLE 6 Results generated by the method according to the invention(carry over prevention). Colon cancer and normal adjacent tissue sampleswere bisulfite treated with bisulfite treatment protocol B resulting inC6 sulfonated uracil containing DNA. Total DNA was measured with the C3quantification assay version B using a calibration curve made by meansof standard B. The table shows the crossing points and the calculatedDNA amount from 2 replicates. Before the measurement of the methylatedDNA with the HeavyMethyl ™ Assay for the TPEF gene version B, thereactions were contaminated with 10,000 copies of the TPEF ampliconcontaining uracil instead of thymine. The table shows the measured CP of2 replicates and the calculated DNA amount. Finally the methylationpercentages (PMR) were calculated by the ratio of methylated DNA andtotal DNA. HeavyMethyl Assay C3 Quantification Assay for the TPEF geneVersion B Version B CP CP ng/PCR CP CP ng/PCR sample type 1^(st) Run2^(nd) Run mean 1^(st) Run 2n^(d) Run mean PMR % standard B   20 ng27.73 27.29 27.63 27.31 standard B   5 ng 29.18 28.58 28.82 28.62standard B   5 ng 29.18 28.89 28.76 28.73 standard B 1.25 ng 31.04 30.7130.57 30.05 standard B 1.25 ng 31.08 30.51 30.33 30.01 standard B 0.31ng 32.9 32.84 31.87 31.46 standard B 0.31 ng 32.78 32.65 32.27 31.69 1normal 33.84 33.49 0.2 28.13 28.16 9.8 2% 2 tumor 37.54 — 0.0 28.5329.14 5.3 0% 3 normal 34.43 34.72 0.2 29.14 29.68 3.0 5% 4 tumor 30.4630.1 1.9 28.23 28.2 9.1 21%  5 normal 34.17 32.89 0.2 28.7 28.92 5.2 4%6 tumor 29.23 28.69 5.6 27.87 27.83 13.1 43%  7 normal 32.97 32.87 0.327.92 28.01 11.7 2% 8 tumor 30.82 30.63 1.3 28.44 28.78 6.4 21%  9normal 33.87 33.55 0.2 28.43 28.59 6.9 3% 10 tumor 30.97 31.19 1.0 28.5829.12 5.2 20%  11 normal 33.14 33.53 0.2 28.11 28.2 9.7 3% 12 tumor33.13 33.66 0.2 27.69 28 13.5 2% 13 normal 33.04 33.57 0.3 28.44 28.866.2 4% 14 tumor 31.08 31.54 0.9 28.03 28.79 8.4 10%  15 normal 33.0933.6 0.2 28.03 28.54 9.0 3% 16 tumor 29.74 29.78 3.0 28.59 28.95 5.554%  17 normal 33.95 33.52 0.2 28.43 28.68 6.7 3% 18 tumor 28.66 28.697.3 28.09 28.09 10.3 70%  19 normal 33.01 34.08 0.2 28.11 28.2 9.7 3% 20tumor 30.03 30.71 1.9 28.19 28.17 9.5 20%  21 normal 36.24 36.98 0.228.92 29.25 4.0 4% 22 tumor 36.04 35.12 0.1 28.58 28.9 5.6 2% 23 normal33.79 33.77 0.2 28.1 28.43 9.0 2% 24 tumor 31.89 32.2 0.5 28.69 29.094.9 10%  neg. contr. — — — — — — —

The results obtained by the standard workflow and the method accordingare compared in a correlation plot (FIG. 5). Every symbol represents asingle sample: quadrates tumor tissues, triangles normal adjacenttissues. The percentage of methylation determined according to thestandard workflow (x-axis) or to the method according to the invention(y-axis) is indicated for each sample.

The exemplary, representative method according to the invention have ledonly in 2 out of 24 samples to a different methylation percentage as thestandard workflow. Although the samples treated according to the methodof the invention were contaminated with uracil containing TPEF ampliconsonly DNA of the samples served as a template for amplification of theTPEF amplicon in nearly all cases. In case of the said two samples, thediffering results occurred presumable because of the low methylationpercentage of the DNA (smaller than 0.2%).

1. A method for providing a decontaminated nucleic acid suitable for DNA5-methylation analysis, comprising: a) contacting a DNA sample with abisulfite reagent under conditions suitable to produce abisulfite-treated sulfonated DNA sample, wherein unmethylated cytosinesof the DNA are sulfonated, sulfonated and deaminated, or comprise amixture of both forms, and wherein said sulfonated, or sulfonated anddeaminated uracil has not been significantly desulfonated; and b)further contacting the bisulfite-treated sulfonated DNA sample with anamount of an enzyme that specifically degrades any contaminatingnonsulfonated-uracil-containing nucleic acids and does not degradesulfonated-uracil-containing nucleic acids thereby providing adecontaminated DNA sample suitable for DNA 5-methylation analysis. 2.The method of claim 1, wherein the contaminatingnonsulfonated-uracil-containing nucleic acids comprisenonsulfonated-uracil-containing carry-over contaminants from a priornucleic acid amplification reaction, and wherein the decontaminated DNAsample is thereby optimized for use in a subsequent amplificationanalysis.
 3. The method of claim 2, wherein the subsequent amplificationanalysis comprises DNA methylation analysis.
 4. The method of claim 1,further comprising: a) mixing the bisulfite-treated sulfonated DNAsample with at least one component required for a polymerase-mediatedamplification reaction or an amplification-based detection assay; b)inactivating, after specifically degrading anynonsulfonated-uracil-containing nucleic acids, the specificallydegrading enzymatic activity; and c) desulfonating, after saidinactivating, the bisulfite-treated sulfonated DNA sample.
 5. The methodof claim 4, wherein inactivating and desulfonating occur simultaneouslyby incubating the mixture at an increased temperature, said incubationsufficient to inactivate the specifically degrading enzymatic activity,and desulfonate the bisulfite-treated sulfonated DNA.
 6. The method ofclaim 4, further comprising, after desulfonating, amplifying thedesulfonated DNA.
 7. The method of claim 4, wherein the specificallydegrading enzyme is at least one selected from the group consisting of aDNA glycosylase and an endonuclease.
 8. The method of claim 7, whereinthe DNA glycosylase is uracil-DNA-glycosylase (UNG).
 9. The method ofclaim 4, further comprising, after inactivating and desulfonating,performing a polymerase-based amplification reaction or anamplification-based assay.
 10. The method of claim 9, wherein thepolymerase-based amplification reaction is initiated by a briefincubation at increased temperature, said incubation being sufficient toinactivate the specifically degrading enzymatic activity.
 11. The methodof claim 9, wherein the polymerase is a heat stable polymerase.
 12. Themethod of claim 9, wherein the polymerase-mediated amplification, or theamplification-based assay is performed in the presence of dUTPs insteadof dTTPs.
 13. The method of claim 1, wherein the amount of thespecifically degrading enzyme is sufficient to degrade substantially allof any nonsulfonated-uracil-containing nucleic acid contaminants anddoes not degrade sulfonated-uracil-contaiing nucleic acids.
 14. A methodfor providing decontaminated nucleic acid suitable for methylationanalysis, comprising bisulfite treatment of a nucleic acid sample toprovide for a sulfonated nucleic acid sample; and contacting thebisulfite-treated sulfonated nucleic acid sample withuracil-DNA-glycosylase (UNG) under conditions suitable to specificallydegrade any contaminating nonsulfonated-uracil-containing nucleic acidsto provide for a decontaminated nucleic acid sample suitable formethylation analysis thereby providing a decontaminated nucleic acidsample suitable for DNA 5-methylation analysis.